text:
Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function.
ABSTRACT Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.
- [Show abstract] [Hide abstract]
ABSTRACT: The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation.Biomolecular NMR Assignments 06/2013; 8(2). · 0.64 Impact Factor -
Article: RME-8 coordinates the WASH complex with the retromer SNX-BAR dimer to control endosomal tubulation.
[Show abstract] [Hide abstract]
ABSTRACT: Retromer is a vital element of the endosomal protein sorting machinery, and comprises two sub-complexes that operate together to sort membrane proteins (cargo) and tubulate membranes. Tubules are formed by the sorting nexin dimer, a key component of which is SNX1. Cargo selection is mediated by the VPS35-VPS29-VPS26 trimer, which additionally recruits the WASH complex via VPS35 binding to the WASH complex subunit FAM21. Loss of WASH complex function leads to dysregulation of endosome tubulation, although it is not clear how this occurs. Here we show that FAM21 also binds to the SNX1-interacting DNAJ protein RME-8. Loss of RME-8 causes altered kinetics of SNX1 membrane association and a pronounced increase in highly branched endosomal tubules. Extending the observations of Popoff et al. (2009), we show that these contain membrane proteins dependent on WASH complex activity for localization to the plasma membrane. We therefore propose that the RME-8/WASH complex interaction provides a potential means to coordinate the activity of the WASH complex with the membrane-tubulating function of the sorting nexins at sites where retromer-mediated endosomal protein sorting occurs.Journal of Cell Science 03/2014; · 5.33 Impact Factor - SourceAvailable from: Nicolas Vitale
Article: Cooperation of MICAL-L1, Syndapin2 and Phosphatidic Acid in Tubular Recycling Endosome Biogenesis.
[Show abstract] [Hide abstract]
ABSTRACT: Endocytic transport necessitates the generation of membrane tubules and their subsequent fission to transport vesicles for sorting of cargo molecules. The endocytic recycling compartment, an array of tubular and vesicular membranes decorated by the Eps15 Homology Domain protein, EHD1, is responsible for receptor and lipid recycling to the plasma membrane. It has been proposed that EHD dimers bind and bend membranes, thus generating recycling endosome (RE) tubules. However, recent studies show that MICAL-L1, a second recently identified RE tubule marker, recruits EHD1 to pre-existing tubules. To date, the mechanisms and events supporting the generation of tubular recycling endosomes remain unclear. Herein, we propose a mechanism for the biogenesis of RE tubules. We demonstrate that MICAL-L1 and the BAR-domain protein Syndapin2 bind to phosphatidic acid, which we identify as a novel lipid component of RE. Our studies demonstrate that direct interactions between these two proteins stabilize their association with membranes, allowing for nucleation of tubules by Syndapin2. Indeed, the presence of phosphatidic acid in liposomes enhances the ability of Syndapin2 to tubulate membranes in vitro. Overall, our results highlight a new role for phosphatidic acid in endocytic recycling, and provide new insights to the mechanisms by which tubular RE are generated.Molecular biology of the cell 04/2013; · 5.98 Impact Factor
Page 1
Rabankyrin-5 interacts with EHD1 and Vps26 to regulate
endocytic trafficking and retromer function
Jing Zhang1, Calliste Reiling1, James B. Reinecke1, Iztok Prislan2, Luis A. Marky2, Paul L.
Sorgen, Naava Naslavsky1,*, and Steve Caplan1,*
1Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of
Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA
2Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha,
Nebraska 68198-5870, USA
Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an
important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for
the recycling regulatory protein EHD1. We have demonstrated this interaction by GST-pulldown,
yeast two-hybrid assay, isothermal calorimetry, and co-immunoprecipitation and found that the
binding occurs between the EH-domain of EHD1 and the NPFED motif of Rank-5. Similar to
EHD1, we found that Rank-5 co-localizes and interacts with components of the retromer complex
such as Vps26, suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of
Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate
receptor (M6PR) transport to the Golgi from endosomes and biosynthetic transport. Moreover,
Rank-5 is required for normal retromer distribution, as over-expression of a wild-type Rank-5-
siRNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of
either Rank-5 or EHD1 impairs secretion of VSV-G. Overall, our data identify a new interaction
between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based
transport and secretion.
retromer; Golgi; biosynthetic transport; secretion; VSV-G
Endocytic trafficking is required for the transport and delivery of membranes and proteins. It
is highly regulated by various proteins, including the C-terminal Eps15 homology domain
(EHD) proteins. There are four mammalian family members, EHD1-4, and a single EHD
ortholog in C.elegans (1) and D. melanogaster (2). EHD proteins are highly homologous and
share 70–86% sequence identity, with EHD1 and EHD3 being the closest paralogs, and all
EHD proteins have been implicated in regulating endocytic transport steps (3). Among
them, EHD1 is the best-characterized, and has a well-established role in regulating recycling
from the endocytic recycling compartment (ERC) to the plasma membrane [(4–7), and
reviewed in (3)]. In addition, EHD1 can influence receptor internalization (8, 9), transport
from pre-sorting endosomes to the ERC (10), and transport from early endosomes to the
*Address correspondence to: Steve Caplan firstname.lastname@example.com and Naava Naslavsky firstname.lastname@example.com, Tel: 402-559-7556
NIH Public Access
Author Manuscript
Traffic. Author manuscript; available in PMC 2013 May 01.
Published in final edited form as:
Traffic. 2012 May ; 13(5): 745–757. doi:10.1111/j.1600-0854.2012.01334.x.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 2
EHD1 and EHD3 also regulate retromer-mediated transport and affect Golgi morphology
(12, 13), although the mechanism for this has remained enigmatic. The retromer controls
retrograde transport from endosomes to the trans-Golgi network (TGN), and is critical for
transport of the cation-independent mannose 6-phosphate receptor (CIMPR) and associated
cargo molecules (14–16). The highly conserved mammalian retromer comprises two
subcomplexes: a dimer of BAR domain-containing sorting nexin (SNX) 1, SNX 2, SNX 5,
and SNX 6, and a heterotrimer composed of vacuolar protein sorting 26 (Vps26) A/B,
Vps35, and Vps29 (17–23). The SNX subcomplex binds to phosphoinositides via Phox
homology (PX) domains and induces membrane tubulation that is dependent on BAR
domains (24–31). The Vps subcomplex is the cargo recognition complex (14), and its
recruitment to membranes is catalyzed by Rab7 and inhibited by the Rab-GAP TBC1D5
(32, 33).
The diverse functions of EHD proteins are facilitated by multiple binding partners. EHD
binding partners almost always contain the tripeptide asparagine-proline-phenyalanine
(NPF), which is a motif that interacts with EHDs through the latter's EH-domain [reviewed
in (3)]. Moreover, recent studies illustrate that an acidic cluster following the NPF motif is
critical for EHD binding selectivity (34, 35). We recently identified a list of potential EHD
binding proteins that contain an NPF motif followed by acidic residues (36). Among these
candidate proteins, we took special interest in Rabankyrin-5 (ANFY1/Rank-5), whose only
known function to date is that of a Rab5 effector involved in macropinocytosis (37). Rank-5
has an N-terminal BTB (Bric-a-brac, Tramtrack and Broad complex)/POZ (POx virus and
Zinc finger) domain followed by 21 successive ankyrin repeats, and a C-terminal FYVE-
finger domain (37–39) that binds to phosphatidylinositol-3-phosphate [PI(3)P]. Rank-5
localizes to early endosomes and macropinosomes, consistent in part with its described role
in coordinating macropinocytosis (37).
In this study, we have identified Rank-5 as a novel binding partner of EHD1. Intriguingly,
we found that Rank-5 co-localizes with and binds to the retromer Vps26 component and
serves to regulate its subcellular localization. Our data support a new role for Rank-5 in the
regulation of retromer-mediated transport and secretion from the Golgi, and provide a
molecular basis for the function of EHD1 and Rank-5 in endosome-to-Golgi transport.
The Rank-5 NPFED motif mediates an interaction with EHD1
To test whether Rank-5 interacts with EHD1, we used GST-fused EH domains of EHD1-4
to pull-down Rank-5 from bovine brain cytosol. As shown in Figure 1A (upper panel),
Rank-5 could be pulled down by GST fused to the EH domain of EHD1 (GST-EH1). On the
other hand, GST-EH3 and GST-EH4 pulled-down barely detectable levels of Rank-5, and
no pull-down was observed by either GST (Control) or GST-EH2. The amount of GST and
GST-fusion proteins used in the pull-down assays is depicted by Coomassie blue staining
(Figure 1A, lower panel). Using yeast two-hybrid analysis, binding of EH1 to Rank-5 was
also detected (Figure 1B, 1st column, −His plate). Moreover, we confirmed the association
of full-length (FL) EHD1 with wild-type (WT) Rank-5 (Figure 1B). Similar to the GST pull-
down results, FL EHD3 exhibited weak binding to Rank-5, whereas no binding was
observed for FL EHD2 and FL EHD4 (Figure 1B).
In previous studies, we and others have demonstrated that C-terminal EHDs selectively bind
to proteins containing NPF motifs that are followed by acidic residues (34, 36). We
therefore hypothesized that mutations in the Rank-5 NPFED motif might interfere with
Rank-5-EHD1 interactions. As shown in Figure 1C, mutating the NPF motif to APA
abolished the binding. Moreover, the binding was significantly decreased by mutating
Zhang et al.
Page 2
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 3
NPFED to NPFAA. These findings indicate that both the NPF-motif and the flanking acidic
residues are required for optimal Rank-5 binding to EHD1.
We further addressed the relevance of the acidic residues (following the NPF motif) by
using Isothermal Titration Calorimetry (ITC) to measure binding affinities of WT Rank-5
NPFED and mutant NPFAA peptides with GST-EH1. We used a single site binding model
to fit each calorimetric binding isotherm. The results of these fits and associated
thermodynamic binding profiles are shown in Table 1. We obtained similar binding
enthalpies of −3.6 kcal/mol (WT) and −4.0 kcal/mol (Mutant) and similar 1:1
stoichiometries (1 mol peptide per mol of protein). However, the Kb for binding of the wild
type peptide was one order of magnitude higher than the Kb of the mutant peptide (Table 1),
resulting in more favorable binding of wild type peptide to protein, by ΔΔG°b = −1.3 kcal/
mol. Table 1 also shows that interaction of both ligands is accompanied with favorable TΔS
contributions. The binding of each peptide is both enthalpy and entropy driven. Favorable
enthalpy contributions correspond to specific van der Waals interactions stabilizing complex
formation, while the favorable entropic contributions includes water and ion releases
overcoming the burial of the surfaces upon complex formation. The less favorable binding
entropy of the mutant peptide is due to less favorable interacting entropy, perhaps because of
its lower release of counterions and water molecules. These data indicate that the ED
residues enhance Rank-5 binding affinity to EH1 significantly.
Rank-5 and EHD1 interact in cells
To determine if Rank-5 binds to EHD1 in vivo, we performed co-immunoprecipitation
analysis. Cells were transfected with either GFP-Rank-5 or GFP vector alone as a control
(Figure 2A–C). Protein expression levels were assessed by immunoblot analysis (Figure
2A), and the effectiveness of immunoprecipitations was demonstrated (Figure 2B). As
shown in Figure 2C, GFP-Rank-5 immunoprecipitated endogenous EHD1 from the total
lysate, whereas the GFP control did not.
Rank-5 has also been observed in association with vesicular structures (37). To determine
whether EHD1 and Rank-5 colocalize, we used specific antibodies that recognize the
endogenous proteins (Figure 2, D–F). Rank-5 partially colocalized with EHD1 on vesicular
structures. These data indicate that Rank-5 and EHD1 might functionally interact in
endocytic pathways.
EHD1 plays a role upstream of Rank-5
We next asked what impact depletion of one binding partner has on the localization of the
other. To address this, we utilized a siRNA approach to impede expression of either Rank-5
or EHD1 in HeLa cells. As depicted, expression of either endogenous Rank-5 (Figure 3A) or
endogenous EHD1 (Figure 3B) was reduced by ~ 90% upon siRNA treatment. To examine
the effect of Rank-5 depletion on EHD1 localization, HeLa cells were transfected with GFP-
Myc-EHD1 (because antibodies to the endogenous protein elicit a relatively weak staining
pattern). No change in the localization of EHD1 was observed upon Rank-5-depletion
(Figure 3, C and D). On the contrary, EHD1-depletion caused a considerably altered
subcellular localization of endogenous Rank-5 from the cell periphery (in Mock-treated
cells; Figure 3E) to the perinuclear area in cells lacking EHD1 (Figure 3F). These data
indicate that EHD1 controls Rank-5 localization and likely functions upstream of Rank-5.
Rank-5 co-localizes with the retromer subunit Vps26
Since both EHD1 and EHD3 have been physically and/or functionally linked to the retromer
complex (12, 13), we hypothesized that Rank-5 might also associate with the retromer. To
determine whether Rank-5 colocalizes with the retromer, HeLa cells were stained with
Zhang et al.
Page 3
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 4
specific antibodies that recognize endogenous Rank-5 and the retromer subunit Vps26
protein. As indicated in Figure 4A–C, Rank-5 and Vps26 displayed partial co-localization
on vesicular structures (see arrows). To determine whether Rank-5 forms a complex with the
retromer, co-immunoprecipitation was performed. HeLa cells were transfected with GFP-
Rank-5 or GFP vector as control, and expression of the proteins was assessed by
immunoblot (Figure 4D). As shown in Figure 4E, GFP-Rank-5 immunoprecipitated
endogenous Vps35 and Vps26 from the lysate, but not SNX2. MICAL-L1, an EHD1
binding partner, was also detected in the immunoprecipitates. These data indicate that
Rank-5 associates with the cargo recognition subcomplex of the retromer, suggesting a
potential role for Rank-5 in the regulation of endosome-to-Golgi transport.
Since the localization of both Rank-5 and the retromer complex subunits are affected by
EHD1-depletion, we next analyzed whether Rank-5 co-localization with Vps26 is impacted
by EHD1 knock-down. Compared to Mock-treated cells (Figure 4A–C and F–H), depletion
of EHD1 led to a 23% increase in the co-localization of Rank-5 and Vps26, mostly in the
perinuclear region (Figure 4, I–K; quantified in L).
To characterize the perinuclear region to which Rank-5 localizes upon EHD1-depletion, we
performed immunostaining with a series of markers (Figure S1). As shown, upon EHD1
knock-down, Rank-5 displayed only very partial overlap with either Giantin (Figure S1, A–
F) or GFP-Rab11 (Figure S1, G–L). Similarly, Vps26 displayed little overlap with GFP-
Rab11 upon EHD1-siRNA treatment (Figure S1, M–R). These results indicate that Rank-5
and Vps26 accumulate in a Golgi proximal region, but display only minor overlap with the
Golgi or ERC upon EHD1-depletion.
Since EHD1 is involved in the regulation of endocytic recycling, we asked whether Rank-5
knock-down might affect the recycling of cargo such as the transferrin receptor (TfR) and
major histocompatibility class I (MHC-I) molecules. HeLa cells were Mock-treated or
treated with Rank-5-siRNA, and assayed for transferrin (Tf) uptake. In Mock-treated cells,
after 5 min of uptake the internalized Tf was still primarily localized to peripheral
endosomes (Figure S2A). On the other hand, in Rank-5-depleted cells, Tf was rapidly
internalized and accumulated in the perinuclear area (Figure S2B). The amount of Tf
internalized measured by flow cytometry displayed a 49% increase in cells depleted of
Rank-5 (Figure S2C). To investigate the effect of Rank-5 depletion on recycling, Mock or
siRNA-treated cells were pulsed with Tf for 5 min and then incubated with complete
medium ("chased") for the indicated time points. As demonstrated, the recycling of Tf was
only modestly delayed in Rank-5 depleted cells (about 14%, Figure S2, D–I).
Similarly, we performed MHC-I uptake and recycling assays in Mock- and Rank-5-siRNA
treated cells. Uptake of MHC-I was accelerated by about 32% in Rank-5 depleted cells
(Figure S3, A–C), and a minor delay in recycling was also observed (Figure S3, D–I). These
data suggest that Rank-5 is involved in clathrin-dependent and -independent internalization
pathways in addition to its role in macropinocytosis (37).
EHD1 and Rank-5 associate with the retromer independently of each other
EHD1 interacts with the retromer and co-immunoprecipitates with Vps26 (12), but whether
this interaction is direct remains unknown. To investigate whether Rank-5 is required for
this interaction, we knocked it down by siRNA (Figure 5A) and then performed co-
immunoprecipitations between EHD1 and Vps26. As shown in Figure 5B, EHD1 remained
associated with Vps26 even upon depletion of Rank-5.
To determine whether EHD1 and Vps26 remain in close proximity in the absence of Rank-5,
HeLa cells were either Mock- or Rank-5-siRNA-treated, and GFP-Myc-EHD1 was
Zhang et al.
Page 4
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 5
expressed in these cells. Cells were then incubated with anti-Myc and anti-Vps26 antibodies
and subjected to a Duolink proximity assay. Protein pairs within ~ 40 nm of each other are
depicted by white dots. EHD1 and MICAL-L1 were used as a positive control (40) (Figure
5, C and D; see white dots), while the GFP vector co-transfected with Vps26 was used as a
negative control and displayed no reaction dots (Figure 5, E and F). As indicated, the close
proximity between EHD1 and Vps26 remained even upon Rank-5-depletion (Figure 5, G–J).
These data suggest that although Rank-5 associates with both EHD1 and Vps26, EHD1
interacts with Vps26 in a Rank-5-independent manner. Similarly, EHD1 depletion by siRNA
treatment (Figure 5K) had little effect on the association between Rank-5 and Vps26 (Figure
5L), suggesting that Rank-5 interacts with Vps26 independently of EHD1.
Depletion of Rank-5 affects localization of Vps26
Given the association of Rank-5 with the retromer, we next asked whether Rank-5
influences the subcellular localization of the retromer complex. In Mock-treated cells, the
retromer complex subunit Vps26 localized primarily to peripheral endosomes, with a pool of
Vps26 co-localized with GM130 (Figure 6, A–C; quantified in G). Upon Rank-5-siRNA
treatment, Vps26 redistributed to the perinuclear region and demonstrated a 31% increased
level of co-localization with GM130 (Figure 6, D–F; quantified in G). Similarly, Rank-5
depletion relocalized SNX2 to the Golgi region, possibly through an indirect mechanism
(data not shown). Rank-5 depletion did induce relocalization of a significant portion of
Rab5-containing endosomes to the perinuclear area (Figure S4). Nevertheless, many Rab5-
containing endosomes were still largely peripheral. Thus while Rank-5 depletion did effect
endosome positioning, its absence affected Vps26-containing vesicles even more
dramatically. Therefore, both endosomal repositioning and potential lack of Vps26-
containing vesicle fusion with the Golgi might account for the observed phenotypes.
To determine whether re-introduction of Rank-5 could rescue the altered Vps26 localization
in Rank-5-depleted cells, we first transfected a siRNA-resistant form of the protein and
examined the effectiveness of its expression by immunoblot. HeLa cells transfected with
WT GFP-Rank-5 exhibited >90% knock-down of both endogenous Rank-5 and GFP-Rank-5
proteins in siRNA-treated cells (Figure 6H, lane 2) compared to Mock (Figure 6H, lane 1).
However, when Rank-5-depleted cells were transfected with the siRNA-resistant-GFP-
Rank-5, both endogenous Rank-5 and GFP-Rank-5 protein levels remained similar to Mock-
treated cells (compare Figure 6H, lanes 3 and 4).
We next transfected the WT siRNA-resistant GFP-Rank-5 into siRNA-treated cells and
examined the localization of Vps26. As shown in Figure 6I–L, transfected cells displayed a
normal pattern of relatively dispersed Vps26 (see cells in yellow border) as opposed to the
Golgi-centralized pattern observed in non-transfected cells lacking Rank-5. 60% rescue in
WT siRNA-resistant Rank-5 transfected cells (Resist-Rank-5) was observed (Figure 6Q). On
the other hand, introduction of a siRNA-resistant Rank-5 mutant lacking a functional NPF
motif (Resist-Rank-5-APA) was unable to rescue the peri-Golgi clustering of Vps26 in cells
lacking Rank-5 (Figure 6, M–P, quantified in Q). These data indicate that Rank-5 and its
binding to EHD1 are required for controlling the localization of Vps26.
Rank-5 depletion affects both the retrieval of M6PR from endosomes to the Golgi and
cathepsin D transport
The retromer complex is required for endosome-to-Golgi transport (14, 16, 31), and an
impaired endosome-to-Golgi axis can lead to delayed biosynthetic transport to late
endosomes and lysosomes (13, 14, 16). We therefore analyzed the effect of Rank-5 knock-
down on endosome-to-Golgi retrieval. HeLa cells grown on cover-slips were treated with
Rank-5-siRNA and subjected to a M6PR antibody uptake assay. We observed a 17%
Zhang et al.
Page 5
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 6
reduction of M6PR at the Golgi in siRNA-treated cells after 30 min of antibody uptake. In
WT siRNA-resistant Rank-5 transfected cells, approximately 60% rescue of M6PR transport
to the Golgi was observed (Figure 7A). Similarly, EHD1- (Figure S5A) or Vps26- (Figure
S5B) depletion reduced the M6PR transport to the Golgi. Consistent with these findings,
there is a significant increase in the subcellular localization of cathepsin D, a lysosomal
lumenal hydrolase, at the Golgi region upon Rank-5-siRNA treatment (Figure 7, C–H,
quantified in B). There is a minor, yet significant corresponding decrease of cathepsin D in
lysosomes upon Rank-5 depletion (Figure S6, A–G).
The AP-1 adaptor complex was also examined because of its involvement in Golgi-to-
endosome transport (41). When Rank-5 was depleted from cells, there was an increase in γ-
adaptin localized to the Golgi area (Figure S7, A–G). However, Rank-5 knock-down had no
obvious effect on Arf1, an important factor in the recruitment of AP-1 to the Golgi (Figure
S7, H–N). Overall, these results demonstrate that Rank-5 plays a role in the endosome-to-
Golgi retrieval and biosynthetic transport from the Golgi.
Rank-5 and EHD1 are required for efficient VSV-G secretion
Since the Golgi is critical for secretion as well as biosynthetic transport, we assessed
whether EHD1 and Rank-5 affect secretion. HeLa cells were either Mock-treated or treated
with either Rank-5-siRNA or EHD1-siRNA. They were then transfected with a temperature-
sensitive mutant of GFP-VSV-G. The cells were incubated at 40°C for 18 h to allow the
accumulation of newly synthesized GFP-VSV-G in the ER. Secretion of VSV-G was then
performed by shifting the cells to 32°C for various time points. As shown in Figure 7I,
secretion of VSV-G was significantly decreased at all time points upon EHD1-knock-down.
Knock-down of Rank-5 also impaired VSV-G secretion at 2 h and 3 h, to a lesser extent than
EHD1-knock-down. Vps26-depletion had little or no effect on VSV-G secretion (data not
shown). These results suggest that EHD1 and Rank-5 are involved in the secretion of VSV-
G, possibly through their effects on normal maintenance of membrane flow to the Golgi.
Recent studies have demonstrated that the selectivity of the C-terminal EH-domain for NPF
motifs is greatly enhanced when the phenylalanine is followed by at least two acidic
residues (34, 36). After searching the databank for proteins containing the motif N-P-F-[D/
E]-[D/E] (36), we chose Rank-5 (N-P-F-E-D) for further investigation because it is a Rab5
effector with a known function in endocytosis/macropinocytosis (37).
We have identified Rank-5 as a novel binding partner of EHD1 in vitro (Figure 1 and Table
1) and in vivo (Figure 2). Moreover, we confirmed that the NPF tripeptide and the
subsequent acidic residues are necessary for optimal binding (Figure 1C and Table 1). Three
other Rab effectors, all containing NPF motifs followed by acidic clusters, have been
reported as EHD-binding proteins: 1) Rabenosyn-5, a divalent Rab4/Rab5 effector that binds
to EHD1 through the first two of its five NPF motifs (42); 2) Rab11-FIP2, the only known
Rab11 effector that contains three NPF motifs (43), binds to both EHD1 and EHD3 (44);
and 3) MICAL-L1, a Rab8a and Rab35 interaction partner that binds EHD1 and recruits it to
tubular membranes (40, 45). These proteins serve as common interaction partners for both
Rab and EHD families, potentially coordinating endocytic transport events (3, 46).
Whereas EHD1 regulates the recycling of cargos from ERC to the plasma membrane [(4–7),
and reviewed in (3)], Rank-5 is known to modulate macropinocytosis (37). However, since
Rank-5 partially co-localized with endosomes, we posited that its binding to EHD1 might
impact endocytic recycling. Surprisingly, knockdown of Rank-5 only moderately delayed
the recycling of Tf and MHC-I, whereas a significant increase in uptake of both Tf and
Zhang et al.
Page 6
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 7
MHC-I was observed (Figure S2 and S3). In EGF-stimulated A431 cells, transferrin uptake
was not significantly altered upon Rank-5-depletion (37), suggesting that under conditions
in which macropinocytosis is not activated, Rank-5 may carry out additional trafficking
Since Rank-5 had only a marginal effect on recycling, we searched for other potential
functions that might be mediated by Rank-5 binding to EHD1. EHD1 and EHD3 regulate
the retromer complex and endosome-to-Golgi retrograde transport (12, 13). Our data
indicate that Rank-5 interacts with the retromer complex Vps26 subunit and Vps35 (Figure
4) and regulates subcellular localization of the complex (Figure 6). EHD1 itself interacts
with the retromer and stabilizes SNX1 tubules (12), and the recruitment of the Vps complex
requires the the GTP binding proteins Rab5 and Rab7 (32, 33). It is likely that through its
interactions with both EHD1 and Rab5 that Rank-5 affects the regulation of retromer
Although EHD1 interacts with the retromer in vivo, it is unclear whether this is by a direct
interaction (12). Our yeast-two hybrid results suggest that there is no direct binding between
either EHD1 and Vps26 or Rank-5 and Vps26 (data not shown). Co-immunoprecipitation
between EHD1 and Vps26 indicates that EHD1 interacts with the retromer complex
independently of Rank-5, since knockdown of the latter has no effect on EHD1-retromer
interactions (Figure 5). Similarly, EHD1 is not required for Rank-5 association with Vps26
(Figure 5). The SNX dimer and Rank-5 bind to PI(3)P-enriched endosomal membranes via
their PX domain (25, 26, 31) and FYVE domain (37), respectively. Our data lead us to
suggest that both EHD1 and Rank-5 indirectly associate with retromer complex subunits,
and the precise mode of this binding will necessitate further study.
The retromer complex is required for endosome-to-TGN transport (14, 16, 31, 47), and
impaired retromer function can lead to delayed biosynthetic transport of lysosomal
hydrolases from the Golgi (13, 14, 16). The functional consequence of Rank-5-depletion on
the retromer was a significant reduction of M6PR retrieval from endosomes and retention of
cathepsin D in the Golgi region (Figure 7). Moreover, Rank-5-depletion led to increased co-
localization of γ-adaptin with the Golgi (Figure S7). This is consistent with the finding that
in EHD3-depleted cells, retrieval of CI-M6PR and recruitment of γ-adaptin are
compromised, leading to impaired transport of cathepsin D from the Golgi (13).
In addition to its effects on endosome-to-Golgi retrieval and biosynthetic transport, both
Rank-5- and EHD1-depletion delayed secretion from the Golgi as determined utilizing a
temperature-sensitive VSV-G mutant (Figure 7I), whereas knock-down of Vps26 had little
effect on VSV-G secretion (data not shown). Indeed, the retromer is also involved in the
retrieval of Wntless to promote the secretion of Wnt proteins (48–53). One potential
explanation for the effects of Rank-5 and EHD1 on secretion is that their depletion impacts
retromer function and thus indirectly affects the maintenance of Golgi membranes and
morphology (13), compromising VSV-G secretion. The delay in VSV-G secretion upon
EHD1 depletion might also be related to the effect of EHD1 on recycling endosomes (54).
Further investigation will be needed to understand the underlying mechanisms.
Overall, we have identified Rank-5 as a novel binding partner of EHD1, both in vitro and in
vivo, and have discerned a new role for Rank-5 in regulating retromer-mediated transport in
an EHD1-dependent manner.
Zhang et al.
Page 7
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 8
Recombinant DNA Constructs
Cloning of the glutathione S-transferase (GST)-EH domains of EHD1-4, and GFP-Myc-
EHD1 have been described previously (4, 40, 42). Human Rabankyrin-5 (Rank-5) was
obtained by PCR from a brain cDNA library (Clontech, Mountain View, CA) and cloned
into TOPO XL vector (Invitrogen, Carlsbad, CA). Rank-5 was subcloned into EGFP-C3
vector and pGADT7 two-hybrid vectors. NPF mutants and siRNA-resistant constructs were
generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Temperature-sensitive GFP-vesicular stomatitis virus glycoprotein (GFP-VSV-G) was
kindly provided by Dr. J. Donaldson (NIH, MD).
Antibodies and Reagents
Affinity-purified rabbit polyclonal antibodies directed against human EHD1 were described
previously (42). Mouse anti-Rank-5 was from Abnova (Taipei City, Taiwan); mouse anti-
actin was from Novus Biologicals (Littleton, CO); mouse anti-SNX2 and mouse anti-
GM130 were from BD Transduction Labs (Franklin Lakes, NJ); sheep anti-TGN46 was
from Serotec (Raleigh, NC); mouse anti-GFP was from Roche Applied Science
(Indianapolis, IN); mouse anti-Cathepsin D was from Santa Cruz (Santa Cruz, CA); rabbit
anti-Giantin, rabbit anti-Vps26 and rabbit anti-Vps35 were from Abcam (Cambridge, MA);
W6/32 antibody to MHC-I (peptide-bound) was obtained from the American Type Culture
Collection (Manassas, VA); mouse anti-M6PR was from BioLegend (San Diego, CA);
mouse anti-VSV-G (clone 8G5FL11) was from KeraFAST (Winston-Salem, NC); Goat anti-
mouse horseradish peroxidase (HRP) and donkey anti-rabbit HRP were obtained from
Jackson ImmunoResearch Laboratories (West Grove, PA); Secondary Cy5-conjugated anti-
mouse, Alexa Fluor 488 or 568 anti-mouse and anti-rabbit were from Molecular Probes
(Eugene, OR).
GST Pulldown and Co-Immunoprecipitation
GST pull-downs were performed as described previously (42). Briefly, purified GST-EH1,
GST-EH2, GST-EH3, GST-EH4 fusion proteins were incubated with glutathione-Sepharose
beads in phosphate-buffered saline (PBS) with 1% Triton X-100 and 0.25mM 4-(2-
aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) for 2 h at 4°C. Beads were
washed four times and incubated with bovine brain cytosol (BBC) at 4°C overnight. Beads
were sedimented and new BBC added and incubated for 4 h. This step was repeated, beads
were washed, and the eluted proteins were separated by SDS-PAGE and immunoblotted
with appropriate antibodies.
For co-immunoprecipitation, transfected cells were lysed in buffer containing 50 mM Tris,
pH 7.4, 150 mM NaCl, 0.5% TX-100, 1.8 mg/ml iodoacetamide and protease inhibitors
cocktail (Calbiochem, Gibbstown, NJ). Protein L-Sepharose beads (Thermo Fisher
Scientific, Rockford, IL) were incubated with anti-GFP antibody for 2 h at 4°C, followed by
three washes with PBS. Lysates were immunoprecipitated with the antibody-bound beads
overnight at 4°C. Immunoprecipitates were washed, eluted, and resolved by SDS-PAGE.
Yeast Two-Hybrid Analysis
The yeast two-hybrid assay was described previously (44). Briefly, Gal4AD and Gal4BD
constructs were co-transformed into the Saccharomyces cerevisiae strain AH109 by the
lithium acetate procedure as described in the instructions for the MATCHMAKER two-
hybrid kit (BD Biosciences Clontech, Palo Alto, CA). The co-transformants were streaked
on plates lacking leucine and tryptophan and incubated at 30°C for 3 days. An average of
four colonies were then chosen and suspended in water. Optical density was measured at
Zhang et al.
Page 8
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 9
600 nm and an equal amount of yeast was spotted on plates lacking leucine and tryptophan
(+HIS) as well as plates also lacking histidine (−HIS). Plates were then incubated at 30°C
until growth of the colonies.
Isothermal Titration Calorimetry (ITC)
Rank-5 wildtype NPF-motif peptide ITVSSDQSVNPFEDVPVV and mutant peptide
ITVSSDQSVNPFAAVPVV were synthesized by GenScript (Piscataway, NJ). The heat for
the reaction of each peptide with the protein was measured directly by isothermal titration
calorimetry using the iTC200 titration calorimeter from Microcal (Northampton, MA). ITC
binding isotherms were obtained as follows; 21 injections of 0.7–2.2 μL peptide (1.2 mM)
were used to titrate a 250 μL protein solution (75 μM) in the reaction cell at 25 °C, the
effective volume of this cell is 200 μL. The reaction heat of each injection is measured by
integration of the area of the injection peak, corrected for the dilution heat of the peptide,
and normalized by the moles of peptide added. The resulting calorimetric binding isotherm,
Heat/(mole of injectant) vs. [peptide]/[protein], were fitted with the Origin 7 software,
supplied by Microcal, Inc. We used a model of one single binding site to fit each binding
isotherms. These fits are characterized by a set of three parameters: binding constant (Kb),
binding enthalpy (ΔHb) and number of sites (n). The Gibbs free energy, ΔG°b were
determined from the equation:ΔGb° = −RTlnKb, where R is the molar gas constant and T is
a temperature at which experiment is performed. The entropy of binding, ΔSb, was
calculated from the Gibbs equation.
Gene Knockdown by siRNA and Rescue using siRNA-Resistant Rank-5 Expression
Oligonucleotide duplexes targeting human Rank-5 or EHD1 (Dharmacon, Lafeyette, CO)
were transfected using oligofectamine (Invitrogen) for 48 h as previously described (42).
The siRNA-resistant rescue construct of Rank-5 was transfected using FuGENE HD (Roche
Applied Science).
Immunofluorescence, Uptake and Recycling Assays, and Quantification
Immunofluorescence staining of HeLa cells was performed as described previously (42, 55).
Images were acquired on an LSM 5 Pascal confocal microscope (Carl Zeiss, Thornwood,
NY) by using a 63X 1.4 numerical aperture objective with appropriate filters. Transferrin
and MHC-I uptake and recycling assays were done as described previously (42).
Quantification of colocalization was done using ImageJ.
Duolink Assay
Duolink kit was purchased from Olink Bioscience (Uppsala, Sweden), and the assay was
done according to the manufacturer's protocol. Briefly, HeLa cells grown on coverslips
were treated with or without Rank-5-siRNA, and transiently transfected with GFP-Myc-
EHD1. The coverslips were then incubated with primary antibodies that bind to the proteins
to be detected: mouse anti-Myc for EHD1 and rabbit anti-Vps26 antibodies. After 1 h of
incubation, the cells were washed in PBS and secondary antibodies conjugated with PLA
probe MINUS and PLUS were added to the reaction and incubated for 1 h at 37°C. The cells
were washed and a ligation solution consisting of two oligonucleotides and the ligase was
added. If the antibodies are in close proximity (< 40 nm), the oligonucleotides will hybridize
to the two PLA probes and join to form a closed circle. The amplification solution consisting
of fluorescently labeled oligonucleotides and polymerase was added, which allows the
signal to be amplified and visualized as a distinct fluorescent spot to be analyzed by
fluorescence microscopy.
Zhang et al.
Page 9
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 10
Anti-M6PR Uptake Assay
HeLa cells were grown on coverslips and treated with or without Rank-5-siRNA. The
coverslips were then washed with PBS, and incubated with anti-M6PR antibody for 30 min
at 37°C in complete medium. Cells were washed and fixed in 4% paraformaldehyde
followed by Golgi staining and the subsequent secondary antibody staining. Quantification
of M6PR at the Golgi was done using LSM Pascal software.
Quantitative Measurements of Uptake by Flow Cytometry
Cells were serum starved in DMEM containing 0.5% bovine serum albumin for 30 min, and
then incubated with Tf-633 (Invitrogen) for a 5 min pulse at 37°C. Cells were washed with
PBS, trypsinized, and transferred to pre-cooled tubes containing 10 ml of ice-cold DMEM,
and pelleted by centrifugation. Cell pellets were fixed in 300 μl of 4% paraformaldehyde. At
least 10,000 cells were analyzed for internalized Tf-633 by flow cytometry analysis (BD
Biosciences, San Jose, CA). For MHC-I uptake, cells were gently dislodged by CellStripper
(Mediatech Inc., Manassas, VA) and washed three times with PBS. Cells were gently rotated
in the presence of W6/32 anti-MHC-I for 1 h, followed by a 20 s acid strip (0.5% acetic
acid, 500 mM NaCl) to remove residual surface-bound antibody (42). Cells were then
washed, fixed, incubated with secondary Cy5-conjugated anti-mouse antibody, and analyzed
by flow cytometry.
VSV-G Trafficking
HeLa cells were grown on coverslips or 35 mm dishes and treated with siRNA for 48 h. A
temperature-sensitive mutant of GFP-VSV-G (56) was transfected in parallel with siRNA.
During the last 18 h, cells were transferred to 40°C to allow accumulation of newly
synthesized GFP-VSV-G in the ER. To measure VSV-G trafficking, cells were pre-
incubated with complete medium supplemented with 20 mM HEPES and 100 μg/ml
cycloheximide (57) for 30 min, and shifted to 32°C for various time points. GFP-VSV-G
reaching the plasma membrane was either visualized by confocal microscopy or measured
by flow cytometry by binding to a mouse anti-VSV-G antibody followed by a secondary
Cy5-conjugated anti-mouse antibody. Secreted VSV-G was calculated at each time point by
dividing surface-expressed VSV-G (Cy5) by overall GFP-VSV-G expressed (GFP), and
subtracting the baseline values at 0 h. Values were normalized in reference to the Mock 3 h
value denoted as 100.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
The authors gratefully acknowledge the support of National Institutes of Health grants R01GM074876 (SC and
NN), and R01GM087455 (SC), the Nebraska Dept. of Health (NN), and P20 RR018759 from the National Center
for Research Resources (SC and NN). This work was also supported by Grant MCB-0616005 from the National
Science Foundation, and a Shared Instrumentation Grant 1S10RR027205 from the National Institutes of Health
(LAM). Partial financial support (to I.P.) from the Slovenian Research Agency (P1 0201) is greatly appreciated.
1. Grant B, Zhang Y, Paupard MC, Lin SX, Hall DH, Hirsh D. Evidence that RME-1, a conserved C.
elegans EH-domain protein, functions in endocytic recycling. Nat Cell Biol. 2001; 3(6):573–579.
[PubMed: 11389442]
Zhang et al.
Page 10
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 11
2. Olswang-Kutz Y, Gertel Y, Benjamin S, Sela O, Pekar O, Arama E, Steller H, Horowitz M, Segal
D. Drosophila Past1 is involved in endocytosis and is required for germline development and
survival of the adult fly. Journal of Cell Science. 2009; 122(Pt 4):471–480. [PubMed: 19174465]
3. Naslavsky N, Caplan S. EHD proteins: key conductors of endocytic transport. Trends in Cell
Biology. 2011; 21(2):122–131. [PubMed: 21067929]
4. Caplan S, Naslavsky N, Hartnell LM, Lodge R, Polishchuk RS, Donaldson JG, Bonifacino JS. A
tubular EHD1-containing compartment involved in the recycling of major histocompatibility
complex class I molecules to the plasma membrane. EMBO J. 2002; 21(11):2557–2567. [PubMed:
5. Guilherme A, Soriano NA, Furcinitti PS, Czech MP. Role of EHD1 and EHBP1 in perinuclear
sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes. Journal of Biological
Chemistry. 2004; 279(38):40062–40075. [PubMed: 15247266]
6. Jovic M, Naslavsky N, Rapaport D, Horowitz M, Caplan S. EHD1 regulates beta1 integrin
endosomal transport: effects on focal adhesions, cell spreading and migration. Journal of Cell
Science. 2007; 120(Pt 5):802–814. [PubMed: 17284518]
7. Lin SX, Grant B, Hirsh D, Maxfield FR. Rme-1 regulates the distribution and function of the
endocytic recycling compartment in mammalian cells. Nat Cell Biol. 2001; 3(6):567–572.
[PubMed: 11389441]
8. Naslavsky N, Rahajeng J, Rapaport D, Horowitz M, Caplan S. EHD1 regulates cholesterol
homeostasis and lipid droplet storage. Biochemical and Biophysical Research Communications.
2007; 357(3):792–799. [PubMed: 17451652]
9. Yap CC, Lasiecka ZM, Caplan S, Winckler B. Alterations of EHD1/EHD4 protein levels interfere
with L1/NgCAM endocytosis in neurons and disrupt axonal targeting. Journal of Neuroscience.
2010; 30(19):6646–6657. [PubMed: 20463227]
10. Cai B, Katafiasz D, Horejsi V, Naslavsky N. Pre-sorting endosomal transport of the GPI-anchored
protein, CD59, is regulated by EHD1. Traffic. 2010; 12(1):102–120. [PubMed: 20961375]
11. Rahajeng J, Giridharan SS, Naslavsky N, Caplan S. Collapsin response mediator protein-2
(Crmp2) regulates trafficking by linking endocytic regulatory proteins to dynein motors. Journal of
Biological Chemistry. 2010; 285(42):31918–31922. [PubMed: 20801876]
12. Gokool S, Tattersall D, Seaman MN. EHD1 interacts with retromer to stabilize SNX1 tubules and
facilitate endosome-to-Golgi retrieval. Traffic. 2007; 8(12):1873–1886. [PubMed: 17868075]
13. Naslavsky N, McKenzie J, Altan-Bonnet N, Sheff D, Caplan S. EHD3 regulates early-endosome-
to-Golgi transport and preserves Golgi morphology. Journal of Cell Science. 2009; 122(Pt 3):389–
400. [PubMed: 19139087]
14. Arighi CN, Hartnell LM, Aguilar RC, Haft CR, Bonifacino JS. Role of the mammalian retromer in
sorting of the cation-independent mannose 6-phosphate receptor. Journal of Cell Biology. 2004;
165(1):123–133. [PubMed: 15078903]
15. McGough IJ, Cullen PJ. Recent advances in retromer biology. Traffic. 2011; 12(8):963–971.
[PubMed: 21463457]
16. Seaman MN. Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer.
Journal of Cell Biology. 2004; 165(1):111–122. [PubMed: 15078902]
17. Bonifacino JS, Hurley JH. Retromer. Current Opinion in Cell Biology. 2008; 20(4):427–436.
[PubMed: 18472259]
18. Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-Golgi network. Nat Rev
Mol Cell Biol. 2006; 7(8):568–579. [PubMed: 16936697]
19. Bugarcic A, Zhe Y, Kerr MC, Griffin J, Collins BM, Teasdale RD. Vps26A and Vps26B subunits
define distinct retromer complexes. Traffic. 2011; 12(12):1759–1773. [PubMed: 21920005]
20. Haft CR, de la Luz Sierra M, Bafford R, Lesniak MA, Barr VA, Taylor SI. Human orthologs of
yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes.
Molecular Biology of the Cell. 2000; 11(12):4105–4116. [PubMed: 11102511]
21. Hierro A, Rojas AL, Rojas R, Murthy N, Effantin G, Kajava AV, Steven AC, Bonifacino JS,
Hurley JH. Functional architecture of the retromer cargo-recognition complex. Nature. 2007;
449(7165):1063–1067. [PubMed: 17891154]
Zhang et al.
Page 11
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 12
22. Kerr MC, Bennetts JS, Simpson F, Thomas EC, Flegg C, Gleeson PA, Wicking C, Teasdale RD. A
novel mammalian retromer component, Vps26B. Traffic. 2005; 6(11):991–1001. [PubMed:
23. Koumandou VL, Klute MJ, Herman EK, Nunez-Miguel R, Dacks JB, Field MC. Evolutionary
reconstruction of the retromer complex and its function in Trypanosoma brucei. Journal of Cell
Science. 2010; 124(Pt 9):1496–1509. [PubMed: 21502137]
24. Carlton JG, Bujny MV, Peter BJ, Oorschot VM, Rutherford A, Arkell RS, Klumperman J,
McMahon HT, Cullen PJ. Sorting nexin-2 is associated with tubular elements of the early
endosome, but is not essential for retromer-mediated endosome-to-TGN transport. Journal of Cell
Science. 2005; 118(Pt 19):4527–4539. [PubMed: 16179610]
25. Cullen PJ, Cozier GE, Banting G, Mellor H. Modular phosphoinositide-binding domains--their role
in signalling and membrane trafficking. Current Biology. 2001; 11(21):R882–893. [PubMed:
26. Merino-Trigo A, Kerr MC, Houghton F, Lindberg A, Mitchell C, Teasdale RD, Gleeson PA.
Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma
membrane following EGF stimulation. Journal of Cell Science. 2004; 117(Pt 26):6413–6424.
[PubMed: 15561769]
27. Seaman MN. Endosome protein sorting: motifs and machinery. Cellular and Molecular Life
Sciences. 2008; 65(18):2842–2858. [PubMed: 18726175]
28. van Weering JR, Verkade P, Cullen PJ. SNX-BAR proteins in phosphoinositide-mediated, tubular-
based endosomal sorting. Seminars in Cell and Developmental Biology. 2010; 21(4):371–380.
[PubMed: 19914387]
29. van Weering JR, Verkade P, Cullen PJ. SNX-BAR-Mediated Endosome Tubulation is Coordinated
with Endosome Maturation. Traffic. 2012; 13(1):94–107. [PubMed: 21973056]
30. Wassmer T, Attar N, Bujny MV, Oakley J, Traer CJ, Cullen PJ. A loss-of-function screen reveals
SNX5 and SNX6 as potential components of the mammalian retromer. Journal of Cell Science.
2007; 120(Pt 1):45–54. [PubMed: 17148574]
31. Carlton J, Bujny M, Peter BJ, Oorschot VM, Rutherford A, Mellor H, Klumperman J, McMahon
HT, Cullen PJ. Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence
sensing of high- curvature membranes and 3-phosphoinositides. Current Biology. 2004; 14(20):
1791–1800. [PubMed: 15498486]
32. Rojas R, van Vlijmen T, Mardones GA, Prabhu Y, Rojas AL, Mohammed S, Heck AJ, Raposo G,
van der Sluijs P, Bonifacino JS. Regulation of retromer recruitment to endosomes by sequential
action of Rab5 and Rab7. Journal of Cell Biology. 2008; 183(3):513–526. [PubMed: 18981234]
33. Seaman MN, Harbour ME, Tattersall D, Read E, Bright N. Membrane recruitment of the cargo-
selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-
GAP TBC1D5. Journal of Cell Science. 2009; 122(Pt 14):2371–2382. [PubMed: 19531583]
34. Henry GD, Corrigan DJ, Dineen JV, Baleja JD. Charge effects in the selection of NPF motifs by
the EH domain of EHD1. Biochemistry. 2010; 49(16):3381–3392. [PubMed: 20329706]
35. Kieken F, Jovic M, Naslavsky N, Caplan S, Sorgen PL. EH domain of EHD1. Journal of
Biomolecular NMR. 2007; 39(4):323–329. [PubMed: 17899392]
36. Kieken F, Sharma M, Jovic M, Giridharan SS, Naslavsky N, Caplan S, Sorgen PL. Mechanism for
the selective interaction of C-terminal Eps15 homology domain proteins with specific Asn-Pro-
Phe-containing partners. Journal of Biological Chemistry. 2010; 285(12):8687–8694. [PubMed:
37. Schnatwinkel C, Christoforidis S, Lindsay MR, Uttenweiler-Joseph S, Wilm M, Parton RG, Zerial
M. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.
PLoS Biol. 2004; 2(9):E261. [PubMed: 15328530]
38. Ito K, Ishii N, Miyashita A, Tominaga K, Kuriyama H, Maruyama H, Shirai M, Naito M, Arakawa
M, Kuwano R. Molecular cloning of a novel 130-kDa cytoplasmic protein, Ankhzn, containing
Ankyrin repeats hooked to a zinc finger motif. Biochemical and Biophysical Research
Communications. 1999; 257(1):206–213. [PubMed: 10092534]
Zhang et al.
Page 12
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 13
39. Kuriyama H, Asakawa S, Minoshima S, Maruyama H, Ishii N, Ito K, Gejyo F, Arakawa M,
Shimizu N, Kuwano R. Characterization and chromosomal mapping of a novel human gene,
ANKHZN. Gene. 2000; 253(2):151–160. [PubMed: 10940552]
40. Sharma M, Giridharan SS, Rahajeng J, Naslavsky N, Caplan S. MICAL-L1 links EHD1 to tubular
recycling endosomes and regulates receptor recycling. Mol Biol Cell. 2009; 20(24):5181–5194.
[PubMed: 19864458]
41. Heilker R, Spiess M, Crottet P. Recognition of sorting signals by clathrin adaptors. Bioessays.
1999; 21(7):558–567. [PubMed: 10472183]
42. Naslavsky N, Boehm M, Backlund PS Jr, Caplan S. Rabenosyn-5 and EHD1 interact and
sequentially regulate protein recycling to the plasma membrane. Mol Biol Cell. 2004; 15(5):2410–
2422. [PubMed: 15020713]
43. Cullis DN, Philip B, Baleja JD, Feig LA. Rab11-FIP2, an adaptor protein connecting cellular
components involved in internalization and recycling of epidermal growth factor receptors. Journal
of Biological Chemistry. 2002; 277(51):49158–49166. [PubMed: 12364336]
44. Naslavsky N, Rahajeng J, Sharma M, Jovic M, Caplan S. Interactions between EHD proteins and
Rab11-FIP2: a role for EHD3 in early endosomal transport. Mol Biol Cell. 2006; 17(1):163–177.
[PubMed: 16251358]
45. Rahajeng J, Panapakkam Giridharan SS, Cai B, Naslavsky N, Caplan S. MICAL-L1 is a Tubular
Endosomal Membrane Hub that Connects Rab35 and Arf6 With Rab8a. Traffic. 2012; 13(1):82–
93. [PubMed: 21951725]
46. Zhang J, Naslavsky N, Caplan S. Rabs and EHDs: alternate modes for traffic control. Bioscience
Reports. 2012; 32(1):17–23. [PubMed: 21981138]
47. Seaman MN. Recycle your receptors with retromer. Trends in Cell Biology. 2005; 15(2):68–75.
[PubMed: 15695093]
48. Belenkaya TY, Wu Y, Tang X, Zhou B, Cheng L, Sharma YV, Yan D, Selva EM, Lin X. The
retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-
Golgi network. Dev Cell. 2008; 14(1):120–131. [PubMed: 18160348]
49. Franch-Marro X, Wendler F, Guidato S, Griffith J, Baena-Lopez A, Itasaki N, Maurice MM,
Vincent JP. Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by
the retromer complex. Nat Cell Biol. 2008; 10(2):170–177. [PubMed: 18193037]
50. Harterink M, Port F, Lorenowicz MJ, McGough IJ, Silhankova M, Betist MC, van Weering JR,
van Heesbeen RG, Middelkoop TC, Basler K, Cullen PJ, Korswagen HC. A SNX3-dependent
retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is
required for Wnt secretion. Nat Cell Biol. 2011; 13(8):914–923. [PubMed: 21725319]
51. Pan CL, Baum PD, Gu M, Jorgensen EM, Clark SG, Garriga G. C. elegans AP-2 and retromer
control Wnt signaling by regulating mig-14/Wntless. Dev Cell. 2008; 14(1):132–139. [PubMed:
52. Port F, Kuster M, Herr P, Furger E, Banziger C, Hausmann G, Basler K. Wingless secretion
promotes and requires retromer-dependent cycling of Wntless. Nat Cell Biol. 2008; 10(2):178–
185. [PubMed: 18193032]
53. Yang PT, Lorenowicz MJ, Silhankova M, Coudreuse DY, Betist MC, Korswagen HC. Wnt
signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells. Dev
Cell. 2008; 14(1):140–147. [PubMed: 18160347]
54. Ang AL, Taguchi T, Francis S, Folsch H, Murrells LJ, Pypaert M, Warren G, Mellman I.
Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma
membrane of MDCK cells. J Cell Biol. 2004; 167(3):531–543. [PubMed: 15534004]
55. Caplan S, Hartnell LM, Aguilar RC, Naslavsky N, Bonifacino JS. Human Vam6p promotes
lysosome clustering and fusion in vivo. J Cell Biol. 2001; 154(1):109–122. [PubMed: 11448994]
56. Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJ, Lippincott-Schwartz J. ER-to-Golgi
transport visualized in living cells. Nature. 1997; 389(6646):81–85. [PubMed: 9288971]
57. Ying M, Grimmer S, Iversen TG, Van Deurs B, Sandvig K. Cholesterol loading induces a block in
the exit of VSVG from the TGN. Traffic. 2003; 4(11):772–784. [PubMed: 14617359]
Zhang et al.
Page 13
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 14
Figure 1. Rank-5 interacts with EHD1 via its NPF-motif
(A) GST-EH-domains of EHD1-4 were used to pull-down Rank-5 from bovine brain cytosol
(BBC). Upper panel shows the Western Blot results; lower panel shows the equal loading of
GST-fusion proteins by Coomassie Blue staining. (B) Yeast two-hybrid interactions between
WT Rank-5 and the EH-domain of EHD1 (EH-1), and full-length EHD1-4. The S.cerevisiae
yeast strain AH109 was co-transformed with the indicated Gal4-binding domain (Gal4BD)
fusion constructs and Gal4-activation domain (Gal4AD)-SV40 (control), together with the
indicated Gal4AD fusion constructs with Gal4BD-p53 (control). Co-transformants were
plated on non-selective (+HIS) and selective (−HIS) media. (C) Yeast two-hybrid interaction
between Rank-5 NPF-motif mutations and EHD1.
Zhang et al.Page 14
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 15
Figure 2. Rank-5 associates with EHD1 in vivo
(A–C) Co-immunoprecipitation of Rank-5 and EHD1. HeLa cells were transfected with
GFP-Rank-5 or GFP, and transfection efficiency was verified by immunoblot (A). After 48
h, cells were lysed and subjected to immuniprecipitation and detection with anti-GFP
antibody (B), or were immunoblotted with anti-EHD1 antibody (C). (D–F) Partial co-
localization between Rank-5 and EHD1. HeLa cells were stained with rabbit anti-EHD1 (D)
and mouse anti-Rank-5 (E) antibodies. Arrows depict representative structures containing
both endogenous proteins. Bar, 10 μm.
Zhang et al.Page 15
Traffic. Author manuscript; available in PMC 2013 May 01.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript


text:
My Wayfair |
See All Trending Items » | |||
New! Our Favorite Renovation Finds » |
New! Contemporary Living Room Photos » |
Home Loft Concept Heavens 18" Embroidered Bird Pillows
Give your home an update with this attractive pillow set. These pillows feature a linen blend cover for soft elegance.
Special Offer:
65 Issues of Us Weekly Magazine
Included with your purchase - a $20 Value (Subscription and Rebate Info)
Home Loft Concept Heavens 18" Embroidered Bird Pillows
Weights & Dimensions
Product Type: Throw pillow | Color: Tan |
Style: Contemporary | Size: Medium (16" - 20") |
Shape: Square | Cover Material: Linen; Other |
Fill Material: Polyester/Polyfill | Lint Free: Yes |
Non-Pilling: Yes | Areas of Support: Back |
Density: Soft | Buttons: Yes |
Applique: Yes | Embroidered: Yes |
Theme: Wildlife | Pattern: Graphic print |
Organic: No | Gender: Unisex |
Life Stage: Adult | Fill Weight: 1 Pounds |
Ply Count: Single-Ply | Tassels Included: No |
Piped Edges: Yes
| Removable Cover: Yes
Pre-Shrunk: No | Washing Method: Dry clean only |
Iron Safe: Yes | Eco-Friendly: No |
Product Care: Do not wash or bleach | Country of Manufacture: China |
General Conformity Certificate: Yes |
About the Manufacturer
Home Loft Concept Heavens 18" Embroidered Bird Pillows
Still have questions about this product? We're here to help
Customer Reviews
5 star | 4 | |
4 star | 1 | |
3 star | 0 | |
2 star | 0 | |
1 star | 0 |
Bright and beautiful Julie from Mountain Lakes, NJ– Verified Buyer These pillows are gorgeous. The bird is done in needlepoint and the color are even more brilliant than in the picture. The head is more of a teal green than the blue in the picture. Looks great against the neutral background. The pillows are very well made with a corded seam and hidden zipper closure. The pillows came in a vacuum sealed back so they were flat when they came. It takes a bit of time and fluffing for them to puff up like they should. |
Painted bunting pillows Barbara from Florida– Verified Buyer I recently purchased a new red sofa and bought these pillows for it. They are gorgeous on it. Well worth the money. |
Cute Chanel from West Bloomfield, MI– Verified Buyer Pillows seemed very thin but puffed out a little after opening. |
Heavens 18" Embroidered Bird Pillows (Set of 2) Carolyn from Atmore, AL– Verified Buyer I love them. They are exactly what I wanted and the colors and workmanship is excellent. |
Very pretty Donna from Fairfax, VA– Verified Buyer Very pretty pillows, embroidery well done and zippers well hidden. Very happy with this purchase. |
Heavens 18" Embroidered Bird Pillows is also sometimes listed under the following manufacturer numbers: 348020

subset: commoncrawl


text:
- Like
- Thanks for voting!
- Download
- Collection
« Previous
Page 1 of 5
Next »
Loading Comments
- This video shows a couple having sexual intercourse while working out.
Select the details below that best describe this video. We appreciate your assistance and will use this information to improve our service to you.
Are you sure you want to remove this video from your favorites?


text:
Cisco Systems
This employer has taken extra steps to respond to reviews and provide job seekers with accurate company information, photos, and reviews. Interested for your company?Learn More.
Employer Engaged
Interview Question
Interview Answer
1 Answer
i took the "i'm too qualified" approach. Not the best way to go.
Add Answers or Comments
To comment on this, Sign In or Sign Up.


text:
Music Director Charles Hazlewood and Singers Sandile Kamle and Pauline Malefane
From the South African production of the opera Carmen, and Yiimimangaliso: The Mysteries, an opera based on the medieval Chester Mystery plays: Music Director Charles Hazlewood and Singers Sandile Kamle and Pauline Malefane. The operas were staged in Londons West End to rave reviews. They are currently making their American premiere at the Spoleto Festival in Charleston, South Carolina that runs May 24-June 9. (www.spoletousa.org). Hazlewood went to South Africa and auditioned over a thousand performers for Carmen. The Mysteries has a 34-member South African cast and is performed in four languages: Afrikaans, Xhosa, Zulu and English. The Mystery plays were popular medieval street theatre performed in the streets of towns all over Europe. They are dramatic versions of stories from the Bible.
Copyright © 2002 NPR. For personal, noncommercial use only. See Terms of Use. For other uses, prior permission required.
Copyright © 2002 NPR. All rights reserved. No quotes from the materials contained herein may be used in any media without attribution to NPR. This transcript is provided for personal, noncommercial use only, pursuant to our Terms of Use. Any other use requires NPR's prior permission. Visit our permissions page for further information.
NPR transcripts are created on a rush deadline by a contractor for NPR, and accuracy and availability may vary. This text may not be in its final form and may be updated or revised in the future. Please be aware that the authoritative record of NPR's programming is the audio.
Related NPR Stories
You must be signed in to leave a comment. Sign In / Register
Please keep your community civil. All comments must follow the NPR.org Community rules and terms of use, and will be moderated prior to posting. NPR reserves the right to use the comments we receive, in whole or in part, and to use the commenter's name and location, in any medium. See also the Terms of Use, Privacy Policy and Community FAQ.


text:
Creepy Monsters, Sleepy Monsters
A Lullaby
From Library Staff
From the critics
Community Activity
Add a CommentThe Creepy Sleepy monsters book is an adorable story about monsters who are preparing to go to sleep and can't. The story begins with a day in a creepy monsters life and how they play and move. Monsters then go home to eat their dinner and take their bath so they can prepare for bedtime. When bedtime comes the monster's are restless and find it difficult to sleep. They fall asleep at the end of the story. My recommendation of the story is that it is suitable for children ages 7and 8 any younger may scare children especially since ages four or five commonly is when children can have nightmares


text:
Today at the REI Outlet, you can score this adorable (and practical) Girl's Columbia June Lake Fleece Jacket for just $13.73, regularly $36. Choose from pink, purple or turquoise. As of the time of this post, all three colors are available in small, … [Read more...]
TERRA Chips Original – $.35 Under 1-Oz Bag, Shipped
This awesome deal is BACK -- if you don't take advantage of it back in January, now is your chance! (And even better, the price has come down a few more pennies.) Amazon has TERRA Original, Sea Salt, 1 Ounce (Pack of 24) for just $8.36 when you … [Read more...]
Ouddy Stainless Steel Fine Mesh Strainers, Set of 3 – $6.39
WOOT! Another awesome kitchen deal from Amazon. (Keep 'em coming!) Get Ouddy Stainless Steel Fine Mesh Strainers, Set of 3 for just $6.39 when you use the coupon code BFB5IK2N at checkout. This set of strainers come in 2 inch, 2.6 inch and 3.2 … [Read more...]
Ouddy Red Silicone Whisk Set (2-Piece Set) – $7.19
Hurry over to Amazon to score a great deal on these Ouddy Silicone Whisk with Stainless Steel Handles, Set of 2. Get them for just $7.19 when you use the coupon code ZF54PT9F at checkout. Clearly with that red silicone, these are meant to be for … [Read more...]
Dry-Erase Magnetic Combination Cork Board — $5.99, Shipped
Amazon has the Quartet Dry-Erase Magnetic Combination Board for only $5.99, regularly $16.50! What an awesome deal on such a useful thing! ** If you don't see the price come up right away, please check on the right under "Other Sellers on Amazon". … [Read more...]
Mets vs. Marlins Tickets for $5 (April 19)
Score $5 tickets to the Mets! The New York Mets are offering a limited number of $5 tickets to their Sunday, April 19th home game against the Miami Marlins. The first pitch will be thrown out at 1:10 pm EST. More details HERE. Note that … [Read more...]
*HOT* Baby Jogger City Lite Stroller — Only $104.99, Shipped (After Gift Card)!
Amazon has this Baby Jogger City Lite Stroller for $179.99 right now, and you'll get a $75 Amazon gift card with your purchase. That's like paying only $104.99 shipped -- which is MUCH lower than the best price on record. To get this deal: go … [Read more...]
Curel Body Lotion – $4.09, Shipped
Another great personal care deal for you! Get Curel Itch Defense Lotion, 13 Ounce for just $4.09 when you click on the $1 off e-coupon and check out thru Subscribe & Save with five active subscriptions in a month. This is a good $2 - $3 … [Read more...]
Aussie Shampoo – 6 Bottles for $8.42, Shipped *HOT*
Oh my gosh, you guys, this is an awesome deal on shampoo! Get Aussie Moist Shampoo 13.5 Fl Oz (Pack of 6) for just $8.42 when you clip the $3 off e-coupon and check out thru Subscribe & Save with five active subscriptions in a month. That's … [Read more...]
{LAST DAY} Dr. Seuss's Birthday Offer | 5 Hardcover Books + Growth Chart for $5.95, Shipped
Today is the last day to take advantage of one of my favorite offers of the year! The Dr. Seuss Book of the Month club is offering the Dr. Seuss birthday package for only $5.95, which includes:
- 5 Classic Dr. Seuss Books: The Cat … [Read more...]


text:
Artist: Jill Scott
Album: Who Is Jill Scott? Words and Sounds Vol. 1
Genre: R&B
{Hello, hello, hello, hello
Hello, hello, hello, hello
Hello, hello, hello, hello
{This morning my man exclusively introduced me
To some good extra lovin'
He was lickin', and suckin' on everything
Just the way he should
This morning's extra lovin' was good
We lay there sweaty, sex funky, happy as we wanna be
Lovin' exclusively, my man and me all night all morning
So our stomachs were growlin', hungrily}
{So I jumped up and rushed to the store
In hopes of coming back and getting a little more of
This morning's good extra lovin'
At the market the people were pushing and shoving
Trying to be the next in line, the turn was mine
The new girl at the counter was cute
But not as fine as me}
{Was this some kind of women's intuition, some kind of insecurity
Nah, 'cuz my man is happy at home lovin' me exclusively
So I shook my head 'wassup'
"Hello" she smiled as she hung me up
"Orange juice, $3.29
Croissants $4.85"
She sniffed, "Butter 89 cents"
She sniffed, "Strawberries a dollar 50 a pint"
She sniffed, and sniffed, and sniffed, and sniffed again
And then replied, "Raheem, right"
Writer(s): Jill H. Scott
Copyright: Jat Cat Music Publishing Inc., Blue'S Baby Music, Universal Music Corp.
Lyrics powered by www.musiXmatch.com
Get the embed code
Note: When you embed the widget in your site, it will match your site's styles (CSS). This is just a preview!
Preview the embedded widget
Jill Scott - Who Is Jill Scott? Words and Sounds Vol. 1 Album Lyrics | |
1. | Gettin' in the Way |
2. | Do You Remember |
3. | Exclusively |
4. | A Long Walk |
5. | I Think It's Better |
6. | He Loves Me (Lyzel In E Flat) |
7. | It's Love |
8. | The Way |
9. | The Roots (Interlude) |
10. | Brotha |
11. | Slowly Surely |
12. | One Is the Magic # |
13. | Watching Me |
14. | Show Me |
15. | Honey Molasses |
16. | Jilltro |
17. | Love Rain |
18. | Try / Love Rain (Remix) |
Jill Scott Lyrics provided by SongLyrics.com
1 | Rightness |
2 | Imagination/Crown Royal Suite |
3 | I Keep/Still Here |
4 | Try / Love Rain (Remix) |
5 | Lovely Day |
6 | I Love You |
Genres |
Top Lyrics |
SongLyrics |
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.


text:
.. and etc. And 'Brazilian Waxes/Landing Strips' on women? Why do people do this? Genuine question. Is it a cleanliness thing? What's wrong with body hair?
DataLounge get your fix of gay gossip, news and pointless bitchery.
Password: [Login]
Click here to create a new authenticated name.
If you have lost your password, click here to have it sent to your email address.
Click here to renew or make a new contribution.
If you have lost your contribution code, click here to have it sent to your email address.
Talking to DataLounge servers.Please wait a moment...
[refresh] [post a new thread]
Create a new thread [back to threadlist]
Keywords (list the proper names of people, places or things in this post.)
[save post]


text:
Canstar Community News - ONLINE EDITION
Marriage commissioners offer custom wedding services
For Chyrle Kyritz, no two wedding services are alike.
"Each of my ceremonies is custom-written," said Kyritz, a wedding commissioner from St. Francois Xavier.
Kyritz became a marriage commissioner 12 years ago after retiring as a high school teacher, and often uses her language skills to help couples find the perfect words for their big day.
She is one of dozens of marriage commissioners licensed by the Manitoba Vital Statistics Agency.
Vital Statistics director Susan Boulter said any permanent resident of Manitoba who's a Canadian citizen and over age 18 can apply to become a marriage commissioner. There is no exam or fee. Individuals must complete an application form which has to be accompanied by a personal resume and three letters of character reference.
A marriage commissioner is able to perform a civil wedding ceremony within the province, but Vital Statistics also issues a permit allowing someone to conduct a ceremony on a one-time basis.
Sanford resident Vernon Henry decided to become a wedding commissioner 15 years ago after providing pulpit relief and witnessing numerous weddings as a member of the United Church.
Henry and Kyritz both say they have found many younger couples lack a religious background and often need guidance when planning their ceremonies.
"I might give them some suggestions," said Henry, who has performed about 40 ceremonies in locations ranging from his home to a farmer's fields.
Lisa Sylvestre is a teacher in Winnipeg who enjoys her summer job performing wedding ceremonies in locations throughout southern Manitoba.
"I love weddings," she said. "I love the romance of them."
With experience in the hospitality industry, Sylvestre sometimes finds herself working with a couple to ensure their ceremony fits with their wedding's theme.
Boulter said about 6,000 weddings take place annually in Manitoba, and a large number of them are held somewhere other than in a church.
"There are fewer and fewer weddings taking place in churches," she said.
Boulter cautioned that unlike in the movies, Manitoba couples can't get married on the spot. They are required to purchase a marriage license from an authorized agent and show identification before signing the license along with the agent.
They must then wait 24 hours before having their wedding service performed and signing a marriage registration document with two witnesses and the person licensed to perform the ceremony. The document is then registered with Vital Statistics.
"We want to make sure that they get everything accurate," she said.
Boulter said she recommends that couples planning a destination wedding outside of Canada make sure to check into the legality of their marriage both in that country and in Manitoba.
The list of Manitoba marriage commissioners in rural Manitoba is available at http://vitalstats.gov.mb.ca/pdf/marriage_commissioners_rural.pdf. Sylvestre's website is http://weddingsmanitoba.com/Home_Page.html. Kyritz can be reached at 204-864-2513 and Henry at 204-228-8428.
Fact Check
Have you found an error, or know of something we've missed in one of our stories?
Please use the form below and let us know.
Having problems with the form?
Contact Us DirectlyMore The Headliner
You can comment on most stories on winnipegfreepress.com. You can also agree or disagree with other comments. All you need to do is be a Winnipeg Free Press print or e-edition subscriber to join the conversation and give your feedback.
You can comment on most stories on winnipegfreepress.com. You can also agree or disagree with other comments. All you need to do is be a Winnipeg Free Press print or e-edition subscriber to join the conversation and give your feedback.
Have Your Say
New to commenting? Check out our Frequently Asked Questions.
The Winnipeg Free Press does not necessarily endorse any of the views posted. By submitting your comment, you agree to our Terms and Conditions. These terms were revised effective April 16, 2010.
- Back to Top
- Return to The Headliner
More The Headliner
(1 of 11 articles for this week)
La Salle residents air their views about development
03/4/2015 1:00 AM 0This Just In
@CanstarHeadline: La Salle Chamber of Commerce is anticipating new members as the community grows. winnipegfreepress.com/our-communitie… #cbn 9:02 am on Mar 5
@LanceWPG: Local entrepreneur lighting up the candle market #cbn - via @WinnipegNews shar.es/1WVXw6 via @ShareThis 12:58 pm on Mar 4
@CanstarHeadline: La Salle's Kelly Arran brings home gold as a forward on Team Manitoba's ringette team at Canada Winter Games. winnipegfreepress.com/our-communitie… #cbn 11:34 am on Mar 4
@CanstarHeadline: The Baie St. Paul bridge is re-opening on Tues., Mar. 3 after repairs are done. winnipegfreepress.com/our-communitie… #cbn 5:03 pm on Mar 3
@TimesWPG: Falcons lacrosse club hope Canada's national summer sport regains popularity in northwest Winnipeg winnipegfreepress.com/our-communitie… #cbn 4:03 pm on Mar 3
Ads by Google


text:
Extremely nice accommodations very near the airport
People were great
Someone told me it was shabby sheik. They got it correct about the shabby part
The staffs are very nice. Concierge service is very attentive .
The health club, pool and breakfast were all terrific
A little bit far from harbor area restaurants but the park-like setting helped offset this.
This hotel definitely is NOT worthy of three stars.
Our door wouldn't close so we were told to slam it as hard as possible. Shuttle is NOT free to airport. Was told our shuttle could only leave on the hour. Shuttle was scheduled by another customer to leave 40 minutes before we were told. Bad hotel!
It was great to have a shuttle to and from the airport. The rooms was comfortable. It was convenient to have a restaurant on site since there were very few restaurants within walking distance.
It offered a limited amount of free wifi and the username and password could only be used on one device at a time which was annoying.
We received confirmation for a garden view room and got a room overlooking the back lot and garbage bins. The hotel changed our room, but we got a smaller bed.
This place is just a gem. It was spotless. ALL the staff were very friendly and helpful. Modern/updated. Great room. Free Bikes (didn't get to use). Free Airport Shuttle. Cozy little bar w/ Free Wifi. Great prices. It was the perfect place to end our stay in NZ.
I loved this place. It worked out perfectly for us, but it is not too close to the city so if you have luggage and if you want to spend the day in Auckland, you will have to figure out where to keep that luggage for the day (and then get back to it). Cab fare via Discount Cab was a flat $35 (a good deal esp. if you get stuck in traffic) and once we were there it was SO easy to relax.
Friendly staff Confort in rooms, specially the bed and pillows Complimentary wifi in public areas
Continental breakfast buffet 22 NZ dollars, too much for only cereal, cheese, bread and some fruits The full breakfast was 27 NZ dollars and it was added, eggs, beans, pancakes, sausages, tomatoes Food is expensive
Here's why:
We're not the seller At KAYAK we don't set prices, so it's not possible for us to guarantee what other companies are selling.
We aggregate tons of data for you Our service lets you quickly and easily compare results from hundreds of travel sites at once. In this sense, KAYAK is a search engine and we don't guarantee prices we find.
Why aren't prices accurate 100% of the time? Prices on airline seats, hotel rooms and car rentals can change frequently. Several people may also be trying to buy the same travel option simultaneously. As a result, you may find on occasion that certain prices are no longer available.
More questions or if you have a bad price to report to us Please send us an email and someone from our team will get back to you promptly.


text:
Family owned since 1953.
747 S Monroe St
Independent Metals Recycling is a Seattle, Washington based metal recycling service.
In 1984, Marty O'Farrell founded Independent Metals in Seattle, Washington and was joined by members of his family. The company goal is to provide customers with quality recycling metal services through professional, prompt pick-up of all scrap metals. Independent Metals supplies six countries including China, Korea, India, Singapore, Japan and the United States.
We recycle the following metals:
• Steel and iron
• Copper
• Electric motors
• Insulated copper wire
• Stainless steel
• Brass
• Aluminum cans
• And more
Call or visit our website to see how we can help!
Category: Recycling
selling my scrap by Oscar - 01/17/2013
I came by there the other day just as they were about to close. These guys made sure I wasn't rushed and help me unload my metals. They even identified for me which each type of wire was and gave me a very good price. I will be back soon!
DexKnows Mobile profile for Independent Metals Recycling in Seattle, WA


text:
Home > News > Community
Published: Friday August 15, 2014 MYT 12:00:00 AM Updated: Friday August 15, 2014 MYT 11:10:29 AM
by sheila sri priyaphotos by syafiq ikhwan
Some 100,000 residents living in PJS3 battle heavy traffic during rush hours because their residential roads were used as an â¿¿escapeâ¿TM route for those who wished to avoid from paying the toll.
SOME 100,000 residents living in PJS3 battle heavy traffic during peak hours because their residential roads are used as a route to bypass tolls.
Residents of Taman Sri Manja and Taman Medan in PJS3 are among the hardest hit by the traffic woes.
Taman Medan Rukun Tetangga secretary ThavaKumar Sellan said motorists had been using the residential roads to avoid the Damansara-Puchong Expressway (LDP) toll near Bandar Sunway.
In recent years, a large number of motorists from Jalan Kelang Lama heading to Puchong and towards the Kesas highway have also been using the PJS3 neighbourhood roads.
"This has led to a surge in vehicles in the high-density neighbourhood, causing bumper-to-bumper jams during the morning and evening rush hours.
"Sometimes, we spend more than an hour to get out of our housing area and it is worst in the evening," he said.
ThavaKumar said an incomplete link next to Pangsapuri Desa Sepakat, Taman Medan, could be completed by cutting through a plot of vacant land nearby to head towards Puchong.
"We heard the Kinrara-Damansara Expressway (Kidex) may pass this area.
"We hope that if the highway were built, it would divert some of the vehicles from our neighbourhood," he said.
Kidex Sdn Bhd confirmed with StarMetro that the highway would pass the Taman Sri Manja neighbourhood.
Based on a statement from Kidex, Jalan PJS1/25 and Jalan PJS 3/40 are currently being used as a main connection for motorists from Petaling Jaya, Jalan Kelang Lama and New Pantai Expressway to access Kesas, Puchong and Kinrara, and vice-versa, leading to congestion on these roads.
It will alleviate the congestion on Jalan PJS3/40 and Jalan PJS1/25.
It is estimated that the traffic will reduce by 18% in Jalan PJS 3/40 and 39% in Jalan PJS1/25 in 2018 once Kidex is open.
Based on the statement, the reduction of congestion at these roads will be beneficial to residents in Taman Medan, Taman Sri Manja and Taman Dato Harun.
Tags / Keywords: Central Region, Family Community, taman sri manja daily jam kidex
Malaysians abroad share how they celebrate our local festivals
8 Incredible food and wine adventures you can do in Australia!
Feyenoord charged over racist behaviour, missiles
MWC2015: HTC's Triple treat
Airlines and airports that get you to your destination on time
Copyright © 1995-2015 Star Publications (M) Bhd (Co No 10894-D)


text:
Lyrics Depot | |
Lyrics » Billy Joel Lyrics » Everybody Loves You Now Lyrics |
Lyrics Depot is your source of lyrics to Everybody Loves You Now by Billy Joel. Please check back for more Billy Joel lyrics. |
Everybody Loves You Now LyricsArtist: Billy JoelAlbum: Songs In The Attic Now you're in the center of the stage Everything revolves on what you do Ah, you are in your prime; you've come of age And you can always have your way somehow But everybody loves you now You can walk away from your mistakes You can turn your back on what you do Just a little smile is all it takes Yeah, you can have your cake and eat it too Loneliness will get to you somehow But everybody loves you now Ah, they all want your white body And they await your reply Ah, but between you and me and the Staten Island ferry So do I All the people want to know your name Soon there will be lines outside your door Feelings do not matter in your game Yeah, 'cause nothing's gonna touch you anymore So your life is only living anyhow And everybody loves you now Close your eyes when you don't want to see Stay at home when you don't want to go Only speak to those who will agree Yeah, and close your mind when you don't want to know You have lost you innocence somehow But everybody loves you now Ah, you know that nothing lasts forever And it's all been done before Ah, but you ain't got the time to go to Cold Spring Harbor No more See how all the people gather 'round Hey, isn't it a thrill to see them crawl? Keep your eyes ahead and don't look down Yeah, and lock yourself inside your sacred wall This is what you wanted; ain't you proud? 'Cause everybody loves you now
Related: |
All lyrics are the property of their respective authors, artists and labels. |


text:
In reading this piece from a Princeton web site which shows the pros and cons of the Internet when it comes to discerning credible information as opposed to non-credible information, this point jumped out at me, "This means that users always have to question the reliability of the available information. If students are not aware and conscious of this problem, they may use and learn wrong facts about anything from historical information to scientific data."
They also point out, "If teachers are prepared to teach students to be critical in their perception of material derived from the network than this characteristic of the Web may not be such a problem, and it may even turn into a plus by enhancing their research skills. Teaching children to be critical of information presented to them, to check sources, and to try to derive information from various sources teaches them that a search for information entails more than just finding a source."
While I will leave you to read what was written in full, it goes to the heart of what we believe as a people happened on 9/11. I will first state, is our anger towards the Bush administration the reason that some tend to believe he caused that attack? I really want those reading this to think hard on that.
On various political lists that I am on, this article has been circulated around in which Senator John F. Kerry stated that Building 7 was deliberately demolished. Instead of taking that article on face value, I went straight to the horse's mouth so to speak. I called up Senator Kerry's office and an aid that I spoke with knew of this article being circulated and he flat out told me that Senator Kerry never opined or stated that Building 7 was deliberately demolished. What he said was that "one wall had to be taken down" He never said that the whole building was intentionally brought down by a controlled demolition.
In circulating that verification response coming from the senator's aid, I invited others to phone the senator's office to hear exactly what I heard. In fact, should you wish to call the senator's office, here is the number, (202) 224-2742. In listening to his aid he sounded angry. Who can blame him when the article misconstrued the words of the Senator according to his aid? I also told him of certain articles making their way around the Internet that the government was behind the shootings at Virginia Tech and if you could hear the reaction of Senator Kerry's aid, he was equally angered over that one.
Just ask yourself is every tragic event that happens a result of a government conspiracy? When that Blue-Angels accident happened, I was ready for someone to come out and say that this government caused that accident. Someone may just do that.
In reading this article, 911 Debunkers Defend War Criminals, Liars, Mass Murderers it reminds me of the tone coming from the Bush administration shortly after 9/11 when Ari Fleisher (Pres. Bush's former Press Secretary) stated, "Watch what you say" I am not a collaborator as the author states since I do believe that President Bush ignored the terrorist threats prior to this attack. Even Richard Clarke a counter-terrorism expert that served under several administrations including President Clinton's was demoted and ultimately fired by the Bush administration.
The reason was that he was trying to bring into focus the threat of Al Qaeda. The Bush administration even ignored the Hart/Rudman report on terrorism. In his interview with Sixty Minutes correspondent, Leslie Stahl, Clarke said of President Bush, "He ignored it. He ignored terrorism for months, when maybe we could have done something to stop 9/11. Maybe. We'll never know."
What I do find astonishing is that several web sites that have posted that President Bush was directly involved in the 9/11 attacks would post this article on their sites, CIA told of al-Qaeda hijack plot before 9/11. This article reports, "FRENCH intelligence services warned their US counterparts, eight months before the attacks of September 11, 2001, that al-Qaeda was planning to hijack a US-bound plane, a media report said today." It goes onto say, "Le Monde said the document, titled "Aircaft hijack plan by radical Islamists," was part of 328 pages of a DGSE file on al-Qaeda leaked to the paper which it said was practically the entirety of the French intelligence services' dossier on the network." Do these sites believe the conspiracy or the ignoring of terrorist threats? That is where critical thinking comes into play.
Now we get to the juicy part. In an email to someone, I wrote, "I have read so many files concerning 9/11 stating that it was planned by the government as well as those that debunk them. I stand by my original column. As you will see, those that have taken time to respond disbelieve me. That is their right to as guaranteed under the first amendment. This is perhaps the best site that debunks various articles written on 9/11 called, Debunking the 9/11 Myths As you will hear in that pod-cast, often those who write of these theories will quote someone who has written of it without going back to the original source. In a court of law, that is called hear say which is often not entered as evidence.
Wait, it gets even better, for sometime, I have been in contact with Controlled Demolitions Incorporated and my source at that company has often said what comments they do give out are often misconstrued. In fact my source said that their words are "often twisted by theorists". She went onto say, "they have had to resort to saying, "no comment"
I have always respected the work done by the Loizeaux family and it angered me when a poster on a political list stated after 9/11 that they were in on this conspiracy. It was then I wrote in response to their post, "What proof do you have?" None was forthcoming from this poster. I am sorry, but you do not throw out such an assertion without backing it up with facts.
Many who believe that 9/11 was a governmental conspiracy have watched the movie "Loose Change" I want you to read this Alternet.org article in which they interviewed a man that took part in the making of that movie as he stated, "Loose Change" happened by accident. The whole thing started out as a fictional screenplay about me and Dylan and another friend of ours finding out 9/11 was an inside job. It started out as a comedic action film with us being chased by the FBI and all that."
Also written in that Alternet.org article is, this point, "What I encourage people to do is go out and research it themselves. We don't ever come out and say that everything we say is 100 percent. We know there are errors in the documentary, and we've actually left them in there so that people discredit us and do the research for themselves -- the B52 (remarked to have flown into the Empire State Building), the use of Wikipedia, things like that. We left them in there so people will want to discredit us and go out and research the events yourself and come up with your own conclusions."
If you truly want to be angry with the direction this administration has taken us, one only has to read that foreclosures have risen by an astonishing 47 percent. This article cited that a record "149,000 foreclosures" took place in March, they go onto say it was "the highest number on record" Another story just broke the other day in which 3 million factory jobs have been lost since 2000.
If that was not bad, this article Flight from U.S. dollar gathers strength. In that article it reports, "The assault against the U.S. dollar intensified yesterday as the greenback slumped to historic lows against currencies as diverse as the U.K. pound and the Polish zloty amid growing conviction among investors' higher returns lie beyond U.S. borders."
1 | 2