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The Life List by Lori Nelson Spielman is a novel with a plot that has been done before: Someone stands to inherit something but must complete a certain task first. In the case of Brett Bohlinger, the only daughter of Elizabeth Bohlinger of Bohlinger Cosmetics, a successful cosmetics company that's worth millions, she must complete 10 things she left uncrossed on a life list she created at the age of 14. That's 20 years ago! Now think about this: What exactly would a 14-year-old want to do with her life, and how would that compare to what a 34-year-old woman would want in her life? Make amends with a friend, okay. Fall in love, sure. But buy a horse? Have a baby, even in the span of one year? And "perform live, on a super big stage"? Seriously? It's crazy that her mother would think that, instead of a nice inheritance just like her two brothers got, she instead must tackle a "to do" list of 10 things she wanted to do with her life 20 years ago. Brett is just as aghast and confused as I was over this whole thing.
Yes, this kind of story has been done, but Spielman has created her own take on this story. She also brings to the table characters we love (like Sanquita), characters we hate (like Andrew) and characters that make us rethink just who we are and how we treat family (like Joad). There's also the scary kid Peter that Brett must tutor, and I'm surprised this menacing young man does not scare Brett so badly that she swears off ever having kids of her own.
Now aside from those characters, there is the main character Brett. I had some trouble figuring this one out. How could a strong, educated woman possibly overlook Andrew's true motives? She knows what kind of man Andrew is, yet she lets it slide when he keeps asking her about her inheritance, even demanding she quote a figure for God's sake, and trying to weasel his way into her mother's company. If I had been in the same room, I would have whispered this little tip to Brett: "He's only interested in your money." I don't know why she keeps ignoring all of her red flags or his actions, even when he acts like such a creep around her. Perhaps she's scared of being homeless because she lives with HIM and has no job or money for a new place (hello? Your mom's house?), or maybe she thinks she must settle for Andrew because how else will she find a guy to marry and have a baby with in one year's time? (And did she really want that creep to be the father of her children?) Also, I had to wonder over certain other mistakes in good judgment Brett makes, such as relying on how a baby reacts to a guy she's dating to see if he'd make a good father (seriously?), and making it her mission to try to help Evil Peter when it's obvious the kid's LSS (love that, by the way!) has escalated to a dangerous level. Also, since her attorney allows Brett a loophole in how long she can stay in her mother's house, she decides that's the new rule, regardless of what it said in her mother's will. And why did she even think it was a good idea to get intimate with her lawyer? I guess she was too vulnerable or fragile to be on top of things or even realize this quest to check off all 10 items on her list before the deadline was making her a little desperate. I had to give her props, though, for standing up to her brother when he questioned her about living at their mother's house. He just didn't see that his sister was WITHOUT the comfort of an inheritance to find a new place to live. That guy was a piece of work, especially with that little comment he made to Brett after she discovered a secret in her mother's diary.
There are some parts of this story that I just fell in love with, though. Some parts of it that made me laugh or cry. It is a good story, despite having been done, and even though Brett was a character that was hard for me to understand, I still enjoyed reading this story. I could never try to complete a life list created 20 years ago and it was interesting to see Brett just try to do that very thing.
After Brett Bohlinger's mother passes away, she is convinced she stands to inherit not only her mother's multimillion-dollar cosmetics company but also a hefty paycheck to boot. But life never works out the way we want it to. Instead of a nice check, Brett is handed a different piece of paper instead: A life list she created 20 years ago. She must complete 10 items on that list in a year's time, or else Brett stands to lose her inheritance. And after being left with nothing, Brett's only chance of regaining her footing is to move forward with her mother's wishes. It's the start of a journey that will turn her life around.
In this utterly charming debut—perfect for fans of Cecelia Ahern's P.S., I Love You and Allison Winn Scotch's Time of My Life—one woman sets out to complete her old list of childhood goals, and finds that her lifelong dreams lead her down a path she never expects.
1. Go to Paris
2. Have a baby, maybe two
3. Fall in love
Brett Bohlinger seems to have it all: a plum job, a spacious loft, an irresistibly handsome boyfriend. All in all, a charmed life. That is, until her beloved mother passes away, leaving behind a will with one big stipulation: In order to receive her inheritance, Brett must first complete the life list of goals she'd written when she was a naïve girl of fourteen. Grief-stricken, Brett can barely make sense of her mother's decision—her childhood dreams don't resemble her ambitions at age thirty-four in the slightest. Some seem impossible. How can she possibly have a relationship with a father who died seven years ago? Other goals (Be an awesome teacher!) would require her to reinvent her entire future. As Brett reluctantly embarks on a perplexing journey in search of her adolescent dreams, one thing becomes clear. Sometimes life's sweetest gifts can be found in the most unexpected places.


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Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.
The secretory pathway of higher eukaryotic cells is composed of the three membrane organelles endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), and Golgi (Bonifacino and Glick, 2004 ; Appenzeller-Herzog and Hauri, 2006 ). Maintenance of these organelles requires a balance of anterograde (secretory) and retrograde vesicular traffic. Anterograde traffic from ER to ERGIC is mediated by coat protein (COP) II vesicles that form at ER exit sites (Aridor et al., 1995 ; Zeuschner et al., 2006 ) and fuse with the ERGIC that consists of a few hundred tubulovesicular membrane clusters in vicinity of ER exit sites (Appenzeller-Herzog and Hauri, 2006 ). Transport from ERGIC to Golgi is mediated by pleomorphic vesicles (Ben-Tekaya et al., 2005 ) that carry COP I (Presley et al., 1997 ; Scales et al., 1997 ), although the mechanism of their formation remains unknown. Retrograde traffic mediated by COP I vesicles can occur from ERGIC or Golgi and recycles membrane proteins that possess either dilysine signals, including ERGIC-53 and KDEL-receptor, or diphenylalanine signals, such as members of the 24 protein family. This rapid COP I-dependent recycling is distinct from the slow Golgi-to-ER recycling of Golgi resident proteins that is COP I independent and can be either constitutive or induced (Storrie, 2005 ).
Major constituents of anterograde and retrograde transport vesicles are transmembrane cargo receptors that mediate protein sorting by linking soluble cargo on the luminal side and coat assembly on the cytoplasmic side. To date, only few cargo receptors have been studied in detail. The polytopic transmembrane protein Erv29p is known to cycle between ER and Golgi in yeast and to operate as a cargo receptor (Belden and Barlowe, 2001 ). Erv29p is required for efficient packaging of the glycosylated α-factor pheromone precursor into COP II vesicles departing from the ER. Maturation of carboxypeptidase Y and proteinase A, but not other secretory proteins such as invertase, also depends on Erv29p (Caldwell et al., 2001 ). In support of the cargo receptor concept, a hydrophobic sorting signal was identified in α-factor that is required for its interaction with Erv29p and efficient transport (Belden and Barlowe, 2001 ; Otte and Barlowe, 2004 ). Erv29p is conserved among eukaryotes and the mammalian orthologue has been designated Surf4 (Reeves and Fried, 1995 ). Although its function is unknown, it is possible that Surf4 has a similar role in ER-to-Golgi transport in mammalian cells given the extent of homology with Erv29p that includes a dilysine retrieval motif.
The best characterized cargo receptor in mammalian cells is the mannose-specific leguminous type lectin ERGIC-53 (Hauri et al., 2000 ; Appenzeller-Herzog and Hauri, 2006 ). ERGIC-53 is a hexameric type I membrane protein in complex with the luminal EF-hand protein MCFD2 (Zhang et al., 2003 ; Nyfeler et al., 2006 ). This cargo receptor complex cycles between ER and ERGIC (Klumperman et al., 1998 ; Nyfeler et al., 2006 ), and it facilitates ER-to-ERGIC transport of the lysosomal enzymes glycoproteins cathepsin C (Vollenweider et al., 1998 ; Nyfeler et al., 2005 ), cathepsin Z (Appenzeller et al., 1999 ), and the blood coagulation factors V and VIII (Nichols et al., 1998 ; Zhang et al., 2003 ). MCFD2 is dispensable for the transport of the lysosomal enzymes, but it required for the transport of factors V and VIII (Nyfeler et al., 2006 ). In the ER, high-mannose cathepsin Z binds to ERGIC-53 by a combined glycan/β-hairpin signal, and it is subsequently released from ERGIC-53 in the ERGIC (Appenzeller-Herzog et al., 2005 ).
Yet another major cargo receptor is Emp24p in yeast. Emp24p is the founding member of the p24 protein family (Kaiser, 2000 ), and it is required for efficient ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins (Schimmoller et al., 1995 ; Muniz et al., 2000 ). It is conceivable that mammalian p24 proteins also operate as cargo receptors although no cargo protein has been identified. Mammalian p24 proteins are localized in the early secretory pathway and rapidly cycle between the ER and Golgi. To achieve their correct targeting within the early secretory pathway they are in a dynamic equilibrium to form homo- and heterodimers with each other (Emery et al., 2000 ; Jenne et al., 2002 ). All p24 family members are type I membrane proteins and share a common structure, with a short cytoplasmic tail containing binding signals for COP I and COP II coat complexes and a luminal domain with potential secretory cargo binding capabilities (Fiedler et al., 1996 ; Sohn et al., 1996 ; Dominguez et al., 1998 ; Muniz et al., 2000 ). Proteomics analysis revealed that p24 family members are major constituents of COP I-coated vesicles (Stamnes et al., 1995 ). Their involvement in COP I vesicle formation was identified in vitro by using liposomes with Golgi-like lipid composition. Liposomes incubated with the cytoplasmic components Arf1, coatomer, and guanosine triphosphate alone are unable to induce vesicle formation unless cytoplasmic domains of p24 family proteins are present (Bremser et al., 1999 ). P24 proteins seem to have some morphogenetic potential. p23 of the p24 family is an essential gene in mammals, and a heterozygous deletion reduces the levels of this protein and other family members, resulting in dilation of Golgi cisternae (Denzel et al., 2000 ). In cell cultures overexpression of p23 leads to its mislocalization to the ER, which causes expansion and clustering of smooth ER membranes. Mislocalization of p23 to the ER also leads to depletion of endogenous p23 from the Golgi, resulting in dispersion of this organelle (Rojo et al., 2000 ).
In the present study, we have characterized human Surf4, and we found it to localize to and cycle in the early secretory pathway similar to ERGIC-53. Surf4 forms multiprotein complexes with ERGIC-53 and p24 family members. Unexpectedly, silencing of Surf4 together with ERGIC-53 or silencing p25 of the p24 protein family disrupted the Golgi apparatus and led to instability of the ERGIC in conjunction with partial dissociation of COP I.
The following mouse monoclonal antibodies were used: G1/93 against ERGIC-53 (Schweizer et al., 1988 ) (ALX-804-602; Alexis, Lausen, Switzerland), A1/182/5 against BAP31 (Klumperman et al., 1998 ) (ALX-804-601; Alexis), G1/133 against giantin (Linstedt and Hauri, 1993 ) (ALX-804-600-C100; Alexis), anti-β-COP (kind gift from Thomas Kreis, University of Geneva, Geneva, Switzerland), GM130 (BD Transduction Laboratories, Lexington, KY), and 12CA5 against the hemagglutinin (HA) epitope. Rabbit polyclonal antibodies against the following proteins were used: KDEL-receptor (Majoul et al., 1998 ; kind gift from H.-D. Söling, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany); Sec31 (Shugrue et al., 1999 ; (kind gift from F. Gorelick, Yale University, New Haven, CT); p23, p24, and p25 (Jenne et al., 2002 ; kind gifts of F. Wieland, University of Heidelberg, Germany); p115 and GM130 (Barroso et al., 1995 ; Nelson et al., 1998 ; kind gift from D. S. Nelson, University of Alabama Medical School, Birmingham, AL); GRASP65 (Sutterlin et al., 2002 ; kind gift from V. Malhotra, Division of Biology University of California, San Diego, CA). Alexa 488-, Alexa 568- (Molecular Probes Europe, Leiden, The Netherlands); and horseradish peroxidase-coupled antibodies (The Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. Polyclonal antibodies against human Surf4 were produced by immunizing rabbits with a recombinant peptide encompassing amino acids 1–60 of Surf4 fused to glutathione transferase (GST). The N-terminal sequence of Surf4 was amplified by polymerase chain reaction (PCR) by using the primers 5′-CAGGATCCCCGGCCAGAACGACCTGATG-3′ and 5′-CGAATTCTTATTACATGTACTGTTTGGGGGAGCTCTC-3′ and cloned into pGEX-5X2 vector via BamHI and EcoRI. The recombinant hybrid protein was expressed in Escherichia coli BL21 and purified by glutathione-Sepharose 4B column chromatography according to the manufacturer's instruction (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The antiserum was affinity-purified by sequential adsorption to Affigel 15 (Bio-Rad, Hercules, CA)-immobilized GST and GST-Surf4 followed by acid elution.
HeLa cells and HeLa cells stably expressing green fluorescent protein (GFP)-ERGIC-53 (Ben-Tekaya et al., 2005 ) were grown in DMEM, supplemented with 10% fetal bovine serum and 1× nonessential amino acids. HepG2 cells and HepG2 cells stably expressing HA-Surf4 (Breuza et al., 2004 ) were grown in minimal essential medium, supplemented with 10% fetal bovine serum. For metabolic labeling and immunoblotting cells were grown in six-well plates. For immunofluorescence microscopy, cells were grown on coverslips in 12-well plates.
Five days after confluence, HepG2 cells were treated with 10 μg/ml brefeldin A (BFA; Epicenter, Madison, WI) for 90 min, and ERGIC membranes were isolated by subcellular fractionation by using Nycodenz gradients (Breuza et al., 2004 ). Fractions of the Nycodenz gradient enriched in the ERGIC marker ERGIC-53 were pooled and diluted five times with phosphate-buffered saline (PBS). The membranes were centrifuged at 100,000 × g for 1 h, followed by solubilization in 25 mM bis-Tris-HCl, pH 7.0, 2% digitonin, and 500 mM 6-amino-caproic acid. The lysates were cleared at 100,000 × g for 1 h, and then they were separated by Blue Native-PAGE (Hunte et al., 2003 ).
Protein complexes separated by Blue Native-PAGE were separated in a second dimension by SDS-PAGE, and proteins were visualized by Colloidal Blue (Invitrogen, Basel, Switzerland). Gel pieces were excised and washed five times for 1 min with 50 μl of 40% n-propanol followed by five washes (1 min each) with 30 μl of 0.2 M NH4HCO3 containing 50% acetonitrile. The gel pieces were dried in a SpeedVac concentrator (Savant, Farmingdale, NY), and they were reswollen in 10 μl of 100 mM NH4HCO3 containing 0.5 μg of modified trypsin (Promega, Madison, WI). Trypsin digestion was performed at 37°C for 18 h. The supernatants were collected, and the gel pieces were extracted with 15 μl of 0.1% formic acid for 5 min, followed by 15 μl of acetonitrile for 1 min. Extraction was repeated twice, and all supernatants were pooled and dried by SpeedVac. For desalting, the peptides were dissolved in 0.1% trifluoroacetic acid (TFA) and adsorbed on C18 ZipTips (P10 size; Millipore, Billerica, MA). The peptides adsorbed on the tips were washed with 0.1% TFA and eluted with 1.5 μl of 80% AcCN, 0.1%TFA, containing 1 μg/μl α-cyano-4-hydroxycinnamic acid (CHCA; Aldrich Chemical, Milwaukee, IL). The eluate (500 nl) was deposited onto anchor spots of a Scout 400-μ m/36 sample support (Bruker Daltonik, Bremen, Germany), and the droplet was left to dry at room temperature. Mass spectra were recorded on a Bruker Scout 26 Reflex III instrument (Bruker Daltonik). The instrument was calibrated with angiotensin II, substance P, bombesin, and ACTH. The peptides were analyzed in reflector mode using delayed ion extraction with a total acceleration voltage of 23 kV. Fifty to 100 single-shot spectra were acquired to improve the signal-to-noise ratio. Spectrum calibration and peak assignment was carried out with the XMASS 5.0 software package provided by the manufacturer. The Mascot search software (http://www.matrixscience.com) was used for protein identification.
HepG2 cells were solubilized in 50 mM Tris-HCl, pH 7.4, 1% digitonin, 150 mM NaCl, 2 mM CaCl2, and protease inhibitors for 1 h at 4°C, followed by centrifugation at 100,000 × g for 1 h. Supernatants were incubated with anti-ERGIC-53 and anti-HA antibodies covalently coupled via dimethyl pimelimidate to protein A-Sepharose beads (Harlow and Lane, 1999 ) or with anti-p23 and anti-p24 antibodies bound to protein A-Sepharose. Beads were washed four times in 50 mM Tris-HCl, pH 7.4, 0.1% digitonin, 150 mM NaCl, and 2 mM CaCl2. Proteins were separated by SDS-PAGE, and then they were transferred to nitrocellulose membranes for immunoblotting.
siRNA oligos were purchased from Eurogentec (Seraing, Belgium) and QIAGEN (Venlo, The Netherlands). Three siRNA oligonucleotides (oligos) were designed against Surf4 and two against p25. siRNA oligos for ERGIC-53 knockdown were described previously (Nyfeler et al., 2006 ). The most efficient siRNA oligo was determined by immunoblotting, and it was chosen for all further experiments. Surf4 was knocked down using 5′-CGUAUAUUUCAACGCCUUCdTdT-3′ as sense and 5′-GAAGGCGUUGAAAUAUACGdTdT-3′ as antisense oligo. P25 was knocked down using 5′-CCUCAGAAUCACAGUGUUAdTdT-3′ as sense and 5′-UAACACUGUGAUUCUGAGGdTdG-3′ as antisense oligo. Nonsilencing control siRNA was purchased from QIAGEN (Basel, Switzerland). The siRNA was used at a final concentration of 5 nM for transfection directly after cell plating using Hiperfect (QIAGEN) according to the manufacturer's instructions. All knockdown experiments were performed 72 h after transfection.
HeLa cells treated with control siRNA, p25 siRNA, or Surf4/ERGIC-53 siRNA were fixed with 3% paraformaldehyde. 0.5% glutaraldehyde in 10 mM PBS, pH 7.4. After washing in PBS, the cells were postfixed in 1% osmium tetroxide. Fixed samples were dehydrated and embedded in Epon 812 resin (Fluka, Buchs, Switzerland). Sections were stained with 6% uranyl acetate and lead acetate, and then they were analyzed with an EM912 Omega EFTEM electron microscope (LEO Electron Microscopy, Oberkochen, Germany).
Cells were fixed in 3% paraformaldehyde, and then they were permeabilized for 5 min in PBS containing 0.2% Triton X-100, 3% bovine serum albumin (BSA), and 20 mM glycine. For the staining with anti-Surf4 0.5% SDS was included in the permeabilization buffer. For the staining with β-COP antibodies, cells were fixed in methanol:acetone. Primary antibodies were added for 30 min in PBS containing 3% BSA and 20 mM glycine. After washing, secondary antibodies were added for 30 min in PBS containing 3% BSA. Cells were embedded in Mowiol, and then they were analyzed by laser scanning confocal microscopy (TCS NT; Leica Microsystems, Wetzlar, Germany). For the quantification of ERGIC clusters and ER exit sites, cells were stained for KDEL-receptor and Sec31, respectively, and costained for giantin. Knockdown cells were chosen based on a dispersed Golgi pattern indicated by giantin. The Golgi area was subtracted from the KDEL-receptor-stained image, and the ERGIC clusters were counted using Image-Pro Plus software (Media Cybernetics, Bethesda, MD). For the quantification of ER exit sites, spots positive for Sec31 were counted. For quantification of β-COP staining, cells were imaged with a charge-coupled device (CCD) camera (SensiCam; PCO Computer Optics, Kelheim, Germany) connected to a Polyvar microscope (VWR, West Chester, PA). The Golgi area was chosen according to giantin staining. The intensity of β-COP and giantin staining in the Golgi area was determined using Image-Pro Plus software.
HeLa cells expressing GFP-ERGIC-53 were treated with sodium butyrate overnight and plated at a density of 4.5 × 104 cells/ml on 18-mm round coverslips, followed by transfection with siRNA. Eighty hours after transfection, living cells were imaged (Ben-Tekaya et al., 2005 ). Images were acquired with a CCD camera (Orca-3CCD; Hamamtsu Photonics, Hamamatsu City, Japan) by using a Lambda DG4 (Sutter Instrument, Novato, CA) for high-speed filter switching. Image-Pro Plus software was used for image recording and processing. Additionally an Edge filter was used to decrease the background signal. Life spans of individual ERGIC clusters were assessed in eight cells for each condition using the automatic tracking tool of Image-Pro Plus. Diameter and intensity filters were used to exclude the Golgi area and to monitor only prominent ERGIC structures. The life span corresponds to the average life span of all ERGIC structures counted per cell. It was measured for no longer than 5 min with image acquisition every 2 s because with these conditions movement of spots could be tracked pixel by pixel. Increasing the analysis time (and imaging interval) resulted in tedious manual tracking, and it gave similar results. Statistical significance (p ≤ 0.05) of the life span between control and knockdown conditions was probed by Student's t test.
Cells were lysed for 1 h at 4°C in PBS containing 1% digitonin, supplemented with protease inhibitors. Lysates were centrifuged at 20,000 × g for 30 min at 4°C. Forty micrograms of protein per lane were separated by SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted sequentially with primary and secondary antibodies, and visualized by enhanced chemiluminescence (GE Healthcare).
HeLa cells were deprived of l-methionine for 20 min, pulsed for 10 min with 100 μCi of [35S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA), and chased for the indicated times in HeLa culture medium containing 10 mM l-methionine. At the end of the chase, the medium was collected and centrifuged for 10 min at 10,000 × g to remove cell debris. For the 0-min chase time, cells of parallel cultures were homogenized in PBS by passing them 10 times through a 25-gauge needle. Five-microliter aliquots of homogenate and media were trichloroacetic acid (TCA)-precipitated, and radioactivity was measured by scintillation counting. Total protein secretion into the medium was normalized to the total counts in cell homogenates at 0-min chase.
HeLa cells grown on 18-mm glass coverslips were incubated in 37°C hypotonic medium (60 mM NaCl, 20 mM HEPES, pH 7.4, and 2.5 mM MgOAc) for 5 min at 37°C. The cells were washed two times in ice-cold PBS, and then they were fixed on ice using 3% paraformaldehyde and processed for immunofluorescence microscopy.
Although discovered quite some time ago, mammalian Surf4 remains largely uncharacterized. Even its subcellular localization is uncertain. N-terminally tagged Surf4 localizes to the ER, whereas C-terminally tagged Surf4 localizes to the Golgi in transfected cells (Reeves and Fried, 1995 ). In contrast, endogenous Surf4 was identified by mass spectrometry in an ERGIC fraction isolated BFA-treated HepG2 cells (Breuza et al., 2004 ). To characterize endogenous Surf4, we prepared polyclonal antibodies to the N-terminal 60 amino acids of Surf4 fused to GST, and we purified them by affinity chromatography. Affinity-purified anti-Surf4 recognized a protein of ~22 kDa on Western blots in reasonable agreement with the Mr of Surf4 deduced from conceptual translation (Supplemental Figure S1A). The specificity of the antibody was confirmed by silencing Surf4 in HeLa cells by using siRNA (Supplemental Figure S3). In control cells, anti-Surf4 gave an immunofluorescence pattern similar to that of the ERGIC marker ERGIC-53, whereas the staining disappeared after siRNA-mediated knockdown of Surf4 without affecting the distribution of ERGIC-53 (Supplemental Figure S1B). These results indicate specificity of our antibodies against Surf4.
To more precisely establish the localization of Surf4, colocalization studies with various organelle markers were performed by immunofluorescence microscopy. Surf4 prominently stained peripheral ERGIC clusters positive for ERGIC-53 (Figure 1A; also see Supplemental Figure S1B) and partially colocalized with the ER marker BAP31 and the Golgi marker giantin (Figure 1A). The predominant localization of endogenous Surf4 in the ERGIC suggests that Surf4 might be a cycling protein of the early secretory pathway. To test this, the distribution of endogenous Surf4 was studied in HeLa cells treated with BFA that is known to accumulate rapidly cycling proteins in the ERGIC (Lippincott-Schwartz et al., 1990 ). Indeed, this treatment concentrated Surf4 in ERGIC-53–positive structures (Figure 1A) supporting the notion that Surf4 is a cycling protein.
Surf4 is a multispanning membrane protein with its C terminus predicted to face the cytosol. The cytosolic tail carries lysine residues in positions −3, −4, and −5 from the C terminus. Two lysines in positions −3 and −4 are known to function as ER targeting signal mediating retrieval (Teasdale and Jackson, 1996 ) or retention (Andersson et al., 1999 ), depending on amino acids in position −1 and −2. We tested the functionality of the lysine motif by mutating the three lysines to serines in hemagglutinin-tagged Surf4 (HA-Surf4SSS). HA-Surf4 localized to ER and ERGIC very much in contrast to HA-Surf4SSS that localized to the Golgi region (Figure 1B). This result is consistent with and explains the Golgi localization of C-terminally tagged Surf4 (Reeves and Fried, 1995 ). In that study, the C-terminal tagging obviously inactivated the dilysine signal, which is known to be position dependent (Teasdale and Jackson, 1996 ). Collectively, our results indicate that Surf4 cycles early in the secretory pathway in a lysine signal-dependent way, similarly to ERGIC-53.
In search of the function of Surf4, we attempted to identify interacting proteins. To maximize such interactions, Surf4 was accumulated in the ERGIC by treating HepG2 cells with BFA. Both nontransfected and HA-Surf4–transfected cells were analyzed. ERGIC membranes were isolated by Nycodenz gradient centrifugation (Breuza et al., 2004 ), and the gradient fractions were analyzed for organelle markers by Western blotting. Surf4 largely codistributed with the ERGIC marker ERGIC-53 (Supplemental Figure S2). The ERGIC fractions were collected, and the membranes were subjected to Blue Native-PAGE. Western blotting revealed that both Surf4 and HA-tagged Surf4 formed protein complexes of ~60 and 232 kDa (Figure 2A). Because HA-Surf4 behaved like endogenous Surf4 on Blue Native gels (Figure 2A), and it was more abundant, some of the further experiments were performed with HA-Surf4. Separation of the protein complexes by SDS-PAGE in a second dimension showed distinct protein spots of 15–37 kDa, which were identified by mass spectrometry as Surf4 and members of the p24 protein family (Figure 2B). This approach also identified the previously described protein complex of p23, p24, p25, and p27 (Fullekrug et al., 1999 ), demonstrating the accuracy of the method (Figure 2B). The 60-kDa complex seems to contain Surf4 and KDEL-receptor, but the possibility of an interaction of the two proteins has not been investigated in the current study.
To confirm the interaction between Surf4 and p24 family members, coimmunoprecipitation experiments were performed. Because the antibody against Surf4 did not immunoprecipitate endogenous Surf4, the HA-tagged protein was studied in transfected HepG2 cells. Figure 2C shows that both anti-p23 and anti-p24 pulled down HA-Surf4. Inversely, anti-HA pulled down both p23 and p24. Surprisingly, a highly specific monoclonal antibody against ERGIC-53, used as (presumed) negative control, also pulled down HA-Surf4 (Figure 2C). This unexpected result was confirmed for endogenous Surf4 in HeLa cells. Anti-ERGIC-53 pulled down Surf4 but not p23 (Figure 2D). We conclude that Surf4 forms hetero-oligomeric complexes with members of the p24 family and in addition interacts with ERGIC-53.
To obtain more insight into the function of Surf4, we took a silencing approach using siRNA (Supplemental Figure S3A and S3B). A knockdown of Surf4 down to 10% in HeLa cells, had no effect on the distribution of organelle markers for ER (unpublished data), ERGIC, and Golgi (Figure 3B and Supplemental Figures S1B and S4A), nor was total secretion of [35S]methonine-labeled proteins impaired 3 d after siRNA transfection (Supplemental Figure S4B). The serendipitous finding of coimmunoprecipation of ERGIC-53 and Surf4 mentioned above led us to test the combined requirement of Surf4 and ERGIC-53. Strikingly, a double knockdown of Surf4 and ERGIC-53 by siRNA disrupted the Golgi apparatus as visualized by staining for giantin (Figure 3A). Quantification showed that 70% of the cells had a dispersed Golgi (Figure 3B). In contrast, a single knockdown of ERGIC-53 down to 10% (Supplemental Figure S3A and S3B) had no effect on Golgi morphology (Figure 3B and Supplemental Figure S4A), consistent with previous knockdown data (Nyfeler et al., 2006 ) and the observation that mislocalization of ERGIC-53 to the ER did not induce changes of the early secretory pathway (Vollenweider et al., 1998 ). Because ERGIC-53 is known to form a complex with the soluble protein MCFD2 and a knockdown of ERGIC-53 leads to secretion of MCFD2 (Nyfeler et al., 2006 ), we wondered whether the Golgi change was due to the lack of MCFD2 rather than ERGIC-53. However, a double knockdown of Surf4 and MCFD2 had no effect on Golgi morphology (unpublished data), strongly suggesting the specific involvement of ERGIC-53 in maintaining normal Golgi structure together with Surf4.
Next, we asked whether the silencing of p24 proteins would also affect Golgi structure. Besides their proposed role as cargo receptors, p24 family members are thought to function as morphogens in the early secretory pathway. Such a function has mainly been derived from overexpression studies (Blum et al., 1999 ; Rojo et al., 2000 ). In addition, the inactivation of one allele of p23 in mice induces structural changes in the Golgi apparatus, and it reduces the levels of p23, p24, and p25 (Denzel et al., 2000 ). p25 is the only p24 family member containing a canonical dilysine signal. Similar to Surf4 and ERGIC-53, inactivation of the dilysine signal in p25 leads to its mislocalization due to inefficient retrieval back to the ER (Emery et al., 2003 ). Although knockdowns of p23 were reported previously (Vetrivel et al., 2007 ), no knockdown experiments have been performed for p25. The known hetero-oligomerization and interdependence of p24 family members complicates such an analysis. Accordingly, we found that a knockdown of p24 reduced p23 levels and vice versa (unpublished data). Depletion of p25 down to 25% (Supplemental Figure S3A and S3B), however, did not affect the protein levels of p24 or p23, which led us to focus on p25 (unpublished data). Strikingly, the knockdown of p25 in HeLa cells induced a change in Golgi morphology that was indistinguishable from that obtained by the Surf4/ERGIC-53 double knockdown (Figure 3A). Ninety percent of the transfected cells showed fragmentation of the Golgi as visualized by immunofluorescence microscopy (Figure 3B). Importantly, the knockdown of p25 did not change the protein levels of Surf4 or ERGIC-53 and vice versa (Supplemental Figure S3C).
Are the changed Golgi structures identical in Surf4/ ERGIC-53 and p25 knockdowns? As a test, we analyzed the silenced cells by transmission electron microscopy. This analysis indicated that under both knockdown conditions the Golgi ribbon was converted to mini-stacks that otherwise looked unchanged. In particular, the cisternae were not swollen and cisternal stacking was intact, suggesting normal cis-trans topology (Figure 3C). Thus, the changes in Golgi morphology induced by a knockdown of Surf4 and ERGIC-53 or p25 are indistinguishable by both light and electron microscopy.
The finding that a double knockdown of Surf4 and ERGIC-53 and a single knockdown of p25 induced a Golgi phenotype was unexpected because all three proteins are mainly associated with the ERGIC, although they also cycle through the Golgi to some extent (Schweizer et al., 1988 ; Dominguez et al., 1998 ; Klumperman et al., 1998 ). Furthermore, a study on the reconstitution of the secretory pathway in a cell-free assay suggests that p25 plays a role in the de novo formation of the ERGIC (Lavoie et al., 1999 ). Based on these findings, we considered the possibility that the knockdowns might also induce changes at the level of the ERGIC. To detect such changes, HeLa cells depleted of Surf4/ERGIC-53 or p25 were double labeled for the ERGIC/cis-Golgi marker KDEL-receptor and the Golgi marker giantin. Peripheral ERGIC structures were quantified in the knockdown cells, which could readily be identified by a dispersed giantin pattern (Figure 4A). Quantification showed that control cells exhibited 490 KDEL-receptor-positive ERGIC structures on average, whereas cells depleted of Surf4/ERGIC-53 had only 230 and cells depleted of p25 only 300 ERGIC structures per cell (Figure 4, A and B). The reduction of KDEL-receptor-positive ERGIC structures was not due to reduced levels of KDEL-receptor (Supplemental Figure S3C). Clearly, both the Surf4/ERGIC-53 knockdown and the p25 knockdown reduced the number of peripheral ERGIC clusters.
ER export activity is known to be modulated by the cargo load (Aridor et al., 1999 ; Guo and Linstedt, 2006 ). Accordingly, the depletion of cargo receptors may impair ER export that would explain the reduction in ERGIC cluster numbers. If true, one would expect that the number of ER exit sites is reduced in parallel. Therefore, we determined the number of ER exit sites labeled by antibodies against the COP II coat protein Sec31 (Figure 5A). P25 knockdown cells showed 400 ER exit sites on average, which was comparable with the 420 ER exit sites counted in control cells (Figure 5B). Surf4/ERGIC-53 knockdowns exhibited a slightly reduced number of 320 ER exit sites (Figure 5B). These numbers show that the reduction of ERGIC clusters is not paralleled by a similar reduction of ER exit sites.
Do the structural changes of ERGIC Golgi impair total protein secretion? We used a pulse-chase approach to address this question. HeLa cells in which p25 or Surf4 together with ERGIC-53 had been silenced, were pulse-labeled with [35S]methionine and the radioactive proteins secreted into the medium during chase were quantified by scintillation counting after TCA precipitation. Figure 5C shows that neither silencing Surf4/ERGIC-53 nor p25 significantly affected total protein secretion after 3 d, although after 4 d the Surf4/ERGIC-53 reduced secretion (Supplemental Figure 4B), implying that the secretion assay is sensitive enough to detect inefficient anterograde protein transport. Because maximal protein silencing was reached already after 3 d of transfection, we conclude that secretion is initially unaffected by the two knockdown conditions.
To study the dynamics of the ERGIC, we used live cell imaging of HeLa cells stably expressing GFP-ERGIC-53. In this cell line GFP-ERGIC-53 behaves like endogenous ERGIC-53 (Ben-Tekaya et al., 2005 ). Strikingly after p25 knockdown, stationary ERGIC structures hovered about in place more actively and disappeared faster than in control cells (Figure 6 and Supplemental Movie 1). Tracking peripheral ERGIC structures revealed that their relative life span was reduced by 35% (Supplemental Figure S5). Thus, the reduction of ERGIC clusters in p25 knockdown cells can, at least in part, be attributed to a shorter half-life. The ERGIC structures in Surf4/ERGIC-53 knockdown cells could not be analyzed in living cells because no acceptable GFP-tagged marker was available to identify the ERGIC-53 in the absence of ERGIC-53. We speculate, however, that the ERGIC structures in Surf4/ERGIC-53 depleted cells would behave similarly.
Collectively, the morphological, biochemical, and live cell imaging results indicate that cargo receptor silencing destabilizes the ERGIC without initial impairment of overall protein secretion.
P24 family members are known to form complexes with the Golgi matrix proteins GM130, GRASP65 and GRASP55 (Barr et al., 2001 ). These matrix proteins are required for normal Golgi morphology. GM130 is a cis-Golgi localized coiled-coil protein targeted to membranes via the peripheral membrane protein GRASP65 (Barr et al., 1997 , 1998 ). It also binds the vesicle tethering factor p115 (Nakamura et al., 1997 ; Nelson et al., 1998 ). GM130 and GRASP65 are key determinants for maintaining Golgi morphology as their knockdown transforms the Golgi ribbon to mini-stacks (Sohda et al., 2005 ; Puthenveedu et al., 2006 ). The knockdowns of p25 and Surf4/ERGIC-53 produced a Golgi phenotype reminiscent of that observed after a knockdown of GM130 and GRASP65 (Sohda et al., 2005 ; Puthenveedu et al., 2006 ). This led us to study the distribution of Golgi matrix proteins in p25- and Surf4/ERGIC-53–depleted cells. Figure 7 clearly shows that GM130, GRASP65 and p115 remained associated with the dispersed Golgi in both p25 and Surf4/ERGIC-53 knockdown cells. We conclude that the morphological changes of the Golgi are unlikely to be due to impaired binding of matrix proteins to Golgi membranes.
Apart from cycling, a common feature of Surf4, ERGIC-53, and p25 is a dilysine ER retention/recycling signal. Because dilysine signals mediate COP I binding and tails of p24 family members are essential for COP I vesicle formation in vitro (Bremser et al., 1999 ) we wondered whether the depletion of the three cycling proteins would affect COP I recruitment. To this end, Surf4 and ERGIC-53 or p25 were silenced in HeLa cells, and the COP I coat subunit β-COP was localized by immunofluorescence microscopy. Strikingly, the overall signal for β-COP was reduced in both knockdown conditions (Figure 8A), which was not due to lower protein levels (Supplemental Figure S3C). The Golgi region identified by giantin showed less prominent staining for β-COP compared with cells treated with control siRNA (Figure 8, A and B). Quantification of the Golgi area revealed that the β-COP signal was reduced by 40% in Surf4/ERGIC-53 and 30% in p25 knockdowns compared with control cells, whereas the signal for giantin remained unchanged (Figure 8, A and B). After 5-min BFA treatment of control cells, 60% of β-COP staining was lost from the Golgi region, indicating β-COP redistribution from the Golgi to the cytosol (Figure 8B). The results indicate that Surf4/ERGIC-53 and p25 are required for COP I recruitment to membranes of the early secretory pathway.
A loss of COP I from Golgi membranes is known to change the structure of this organelle to the extent that it rapidly tubulates and fuses with the ER. Such an outcome is well known for cells treated with the fungal metabolite BFA. The Golgi changes induced by silencing Surf4 and ERGIC-53 or p25 are clearly different from those induced by BFA. We wondered whether knockdown cells would respond normally to BFA. Figure 9D shows that a 30-min BFA treatment of control cells induced an almost complete disappearance of the Golgi. As expected, giantin showed an ER-like pattern and GM130 redistributed to the ERGIC (Figure 9, A and D). In contrast, after Surf4/ERGIC-53 or p25 silencing, GM130 and p115 were largely resistant to BFA, and they remained in the juxtanuclear area (Figure 9, A–C), very much in contrast to KDEL-receptor that redistributed to the ERGIC (Figure 9A) and the two Golgi markers giantin and GPP130, which redistributed to the ER (Figure 9D).
Obviously, COP I dissociation induced by cargo receptor silencing does not result in a BFA-like effect. Thus, COP I dissociation cannot explain the absence of tubulation of the cis-Golgi. Together with the partial resistance of the cis-Golgi to BFA after cargo receptor silencing, the lack of tubules implies that cargo receptors are required for efficient tubulation. The role of cargo receptors in promoting tubulation of the cis-Golgi indicated by GM130 was assessed by subjecting cells depleted of cargo receptors to hypotonic stress known to cause tubulation of the Golgi (Lee and Linstedt, 1999 ). Cells depleted of Surf4/ERGIC-53 or p25 showed no tubulation of the cis-Golgi, whereas in control cells the cis-Golgi was extensively tubulated after hypotonic stress (Figure 10).
Collectively, these data indicate that silencing Surf4 together with ERGIC-53 or silencing p25 leads to partial dissociation of COP I. Moreover, the partial resistance of the cis-Golgi to BFA and the lack of tubulation after cargo receptor silencing imply that cargo receptors are required for efficient tubulation of the cis-Golgi.
In this study, we characterized human Surf4, and we found it to be associated with the ERGIC and to cycle in the early secretory pathway in a dilysine signal-dependent manner. Erv29p, the yeast orthologue of Surf4, acts as a cargo receptor for glycosylated α-factor in yeast (Belden and Barlowe, 2001 ; Otte and Barlowe, 2004 ). Although a knockdown of Surf4 had no effect on total protein secretion, it remains possible that human Surf4 also operates as a cargo receptor for a limited set of proteins that would not be apparent in a global secretion assay. Previous studies have also implicated Erv29p in ER quality control. In yeast cells lacking Erv29p, misfolded soluble proteins are stabilized, and it was proposed that efficient degradation of these misfolded proteins requires transport between ER and Golgi mediated by Erv29p (Caldwell et al., 2001 ). We found no equivalent function for human Surf4. An efficient knockdown of Surf4 had no effect on the degradation of the Z mutant of α1-antitrypsin a prototype ERAD substrate (data not shown). This observation argues against a general role of Surf4 in ER degradation of misfolded soluble proteins as suggested for Erv29p.
The characterization of Surf4-interacting proteins uncovered a novel role of cargo receptors in maintaining the architecture of ERGIC and Golgi. Surf4 was found to form at least two protein complexes, one complex that has an Mr of 232 kDa and comprises p23, p24, and p25; and another complex of ~60 kDa, which was not further characterized but may contain KDEL-receptor. The serendipitous finding of a coimmunoprecipitation of Surf4 and ERGIC-53 suggests the existence of a third complex. Because ERGIC-53 forms homohexamers (Schweizer et al., 1988 ), this complex can be expected to be very large so that it may not have entered the Blue Native gel. It is widely recognized that p24 family proteins form heterooligomeric complexes with one another, which complicates the functional analysis of these proteins (Dominguez et al., 1998 ). The current study suggests that the situation is even more complex. The major known cargo receptors can form various protein complexes with one another with functional implications for organelle maintenance. Although this was unexpected, an even greater surprise was the observation that a double knockdown of Surf4/ERGIC-53 and a single knockdown of p25 resulted in an identical Golgi and ERGIC phenotype, particularly because the Surf4/ERGIC-53 knockdown did not affect p25 levels and vice versa. There are no indications, however, for a major difference of the phenotypes resulting from the two different knockdowns, neither at the light nor at the ultrastructural level. The phenotype is characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus whereby the Golgi elements were not randomly distributed in the cytoplasm but largely remained in the original area of the initially compact Golgi.
Numerous situations are known in which the Golgi assumes a fragmented phenotype. How do these phenotypes compare with that observed in the present study? The classical phenotype of dispersed Golgi is due to disruption of microtubules by microtubule-active drugs, such as nocodazole. By contrast, silencing of Surf4/ERGIC-53 or p25 had no effect on microtubules (unpublished data) and the Golgi mini-stacks were not randomly distributed in the cytoplasm as in nocodazole-treated cells. Some other knockdown conditions can lead to Golgi fragmentation similar to that described here, although effects on the ERGIC have not been studied. For example, silencing the SNARE protein syntaxin 5 results in Golgi fragmentation that barely affects anterograde transport of VSV-G, but the underlying mechanism is unknown (Suga et al., 2005 ). Silencing of KAP3, the nonmotor subunit of kinesin 2, also results in fragmentation of the Golgi (Stauber et al., 2006 ). Again, anterograde secretory traffic is unaffected, but KDEL-receptor–dependent retrograde transport is abrogated, presumably due to an unexplained redistribution of the KDEL-receptor to the ER. Thus, this phenotype is different. Yet another type of Golgi fragmentation results from silencing golgin-84 (Diao et al., 2003 ). However, this phenotype is accompanied by changes of the ER, and it has been attributed to a defect in anterograde trafficking. Comparing all the known Golgi fragmentation phenotypes, the Golgi phenotype induced by cargo receptor silencing is strikingly similar to that recently reported for knockdowns of the Golgi matrix proteins GM130 and GRASP65 (Puthenveedu et al., 2006 ). Either knockdown prevents lateral linking of Golgi stacks resulting in mini-stacks. GM130 mediates stabilization and targeting of GRASP65, and the two proteins are required for Golgi ribbon formation. As a further similarity to the current work, secretory transport is independent of GM130-mediated Golgi ribbon formation (Puthenveedu et al., 2006 ). Importantly, however, there was no indication of dissociation of GM130 or GRASP65 in cargo receptor knockdowns in the current study, indicating that these two Golgi matrix proteins are not sufficient for Golgi ribbon formation. Moreover, a knockdown of GM130 has no effect on the stability of the ERGIC (our unpublished observations).
Reduced COP I binding for both knockdowns of Surf4/ERIGC-53 and p25 provided a mechanistic explanation for at least some aspects of the phenotype. There are two major different functions of COP I: vesicle formation and stabilization of membranes (Klausner et al., 1992 ; Rothman, 1994 ; Storrie, 2005 ; Bethune et al., 2006a ). COP I vesicles mediate membrane traffic within the Golgi, from cis-Golgi to ERGIC, and from ERGIC to ER. Some rapidly cycling transmembrane proteins are actively recruited to retrograde vesicles by a dilysine signal of their cytosolic tail that directly interacts with COP I subunits (Jackson et al., 1990 ; Cosson and Letourneur, 1994 ; Bethune et al., 2006a ). Surf4, ERGIC-53, and p25 contain such a dilysine signal that is functional in all three proteins (Itin et al., 1995 ; Emery et al., 2003 ; this study). In vitro, the formation of COP I vesicles requires the presence of the cytoplasmic domains of p24 family proteins (Bremser et al., 1999 ). Thus, COP I dissociation from cis-Golgi and ERGIC observed in the current study renders retrograde traffic less efficient. Because anterograde secretory traffic is unaffected this obviously leads to a shortage of ERGIC membranes, which would explain the reduced number and perhaps also shortened life span of ERGIC clusters. For such an outcome with reduced ERGIC-53 cluster numbers one would have to also postulate that in the knockdown cells ERGIC-to-ER transport, although reduced, is slightly more efficient than cis-Golgi to ERGIC transport. This is plausible in view of the proximity of ERGIC and ER, but it cannot be assessed experimentally with current technology.
A function of COP I in membrane stabilization is known from experiments with BFA. On BFA treatment, COP I dissociates from Golgi membranes, and these membranes rapidly tubulate and fuse with the ER. Obviously, COP I protects membranes from tubulation and thereby guarantees organelle integrity and identity. Importantly, neither silencing Surf4/ERGIC-53 nor p25-induced Golgi tubulation despite considerable dissociation of COP I. Under these knockdown conditions COP I dissociation can be assumed to occur at the level of the ERGIC and cis-Golgi, the recycling sites of these cargo receptors. In contrast, overexpression of p25 containing an inactivated dilysine signal does not affect COP I distribution or induce fragmentation of the Golgi apparatus, although it mislocalizes p24 family members to the cell surface (Emery et al., 2003 ). Inversely, the depletion of p25 did not lead to mislocalization of endogenous p24 to the cell surface (unpublished data). Obviously, overexpression of mutated p25 does not impair the function of p25 to the same extent as a knockdown of p25.
Clearly, COP I dissociation induced by cargo receptor silencing does not result in a BFA-like effect. Thus, COP I depletion cannot explain the absence of tubulation of the cis-Golgi. Together with the partial resistance of the cis-Golgi to BFA after cargo receptor silencing, the lack of tubules implies that cargo receptors are required for efficient tubulation. A likely scenario is that cargo receptor tails mediate the interaction of cis-Golgi membranes with microtubules. Microtubules are required for BFA-induced tubulation of Golgi membranes after COP I dissociation and their subsequent consumption by the ER (Lippincott-Schwartz et al., 1990 ). Receptor tails may recruit kinesine-type motor proteins, such as kinesin II (Stauber et al., 2006 ), in the absence of protective COP I coats. Consistent with such a mechanism, the tubulation of anterograde transport intermediates also depends on cargo receptor tails as microinjection of cytosolic tails of p23 and p24 efficiently inhibits tubule formation (Simpson et al., 2006 ). Obviously, p24 and presumably other cargo receptor tails have an inherent tubulation potential which needs to be controlled by COP I coats to maintain Golgi integrity.
Is the Golgi fragmentation in Surf4/ERGIC-53 or p25 knockdown cells due to COP I dissociation? The close similarity of phenotypes resulting from matrix or cargo receptor knockdowns raises the question of whether an interaction of the two classes of proteins is required for maintaining the Golgi ribbon. If so, a knockdown of either protein class would cause an identical Golgi mini-stack phenotype. Such a notion is not entirely hypothetical because p23, p24, and p25 have been reported to be in a complex with GRASP65, GRASP55, and GM130 in vivo and purified GRASPs directly bind to cytoplasmic tails of p24s (Barr et al., 2001 ). In contrast to these observations, we have not seen an interaction of p25, Surf4, or ERGIC-53 with GM130 in immunoprecipitation experiments with antibodies to GM130 (data not shown). Thus, more detailed studies will be required to assess a putative dual interaction of cargo receptors with COP I and matrix proteins. It is worth noting, however, that the ERGIC phenotype induced by cargo receptor silencing is unlikely to be due to impaired matrix/tail interactions, because GM130 is primarily associated with the first Golgi cisterna at steady state (Nakamura et al., 1995 ; Taguchi et al., 2003 ) and is not detectable in the ERGIC (Figure 7). An alternative possibility to explain the Golgi phenotype induced by receptor silencing is a disturbed balance of the amount of Golgi membranes and matrix proteins. Reduced retrograde traffic from cis-Golgi to ERGIC may result in an increase in Golgi membranes without a corresponding increase in matrix proteins, which may affect Golgi ribbon maintenance.
Why does a single knockdown of Surf4 or ERGIC-53 not change Golgi morphology, whereas p25 does? Currently, we can only speculate about the underlying mechanism. One possibility is that the individual levels of ERGIC-53 and Surf4 in the cis-Golgi are lower than those of p25; therefore, only a combined knockdown of Surf4 and ERGIC-53 leads to sufficient dissociation of COP I from the cis-Golgi. Although no information for Surf4 is available, the levels of ERGIC-53 in the cis-Golgi are indeed low, because the recycling of ERGIC-53 between ERGIC and ER largely bypasses the cis-Golgi (Klumperman et al., 1998 ; Ben-Tekaya et al., 2005 ). Alternatively, p25 may not act in isolation because it forms complexes with other p24 proteins that are known to interact with COP I coats via a diphenylalanine rather than a dilysine signal (Bethune et al., 2006a,b ). By indirectly affecting other p24 family members, silencing of p25 may have a greater impact.
In conclusion, we propose the following model for the changes of the early secretory pathway induced by the depletion of Surf4/ERGIC-53 or p25 (Figure 11). The reduction of cargo receptor tails reduces COP I binding to cis-Golgi and ERGIC and impairs retrograde vesicular traffic. Because anterograde traffic is unchanged this defect results in fewer ERGIC clusters. The reduction of cargo receptors in the cis-Golgi also leads to Golgi mini-stacks either due to insufficient cross-linking of cargo receptor tails with Golgi matrix or due to an imbalance of Golgi membranes and Golgi matrix. According to the maturation model, mini-stack formation would start at the cis-Golgi and gradually be completed as the first cis-Golgi cisterna moves and matures in cis-to-trans direction. Whatever the precise mechanism, the current study shows that networks of established and putative cargo receptors are required to maintain the architecture of ERGIC and Golgi. Thus, cargo receptors of the early secretory pathway can have multiple functions by operating both individually and in concert with one another. This striking dual mode of operation will have to be taken into consideration in future attempts to understand the organization and function of the secretory pathway.
We thank Anne Spang for helpful suggestion; Käthy Bucher for technical assistance; Paul Jenö for mass spectrometry analysis; Ursula Sauder for electron microscopy analysis; Adam Linstedt for anti-giantin; David S. Nelson for anti-p115 and anti-GM130; Felix Wieland for antibodies to p23, p24, and p25; Fred Gorelick for anti-Sec31; and Vivek Malhotra for anti-GRASP65. This work was supported by the Swiss National Science Foundation and the University of Basel.
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-10-0989) on February 20, 2008.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.

subset: commoncrawl


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Talking "Out of the Furnace" with Christian Bale, Woody Harrelson, Casey Affleck, Zoe Saldana and Director Scott Cooper
"Out of the Furnace" may be this generation's movie that shows a family's struggle from the ill effects of the Iraq war and the Great Recession.
Director Scott Cooper's powerful drama has an all-star cast that included Christian Bale, Woody Harrelson, Casey Affleck, Willem Dafoe, Forest Whitaker and Zoe Saldana. There's no question the acting is nearly flawless with this cast.
The revenge film is about when a younger brother mysteriously disappears in the Appalachian Mountains after a bare knuckle fight. His older brother takes the matter into his own hands for justice.
Latino-Review was present at the press conference late last month to discuss about the film.
Questions asked from the press ranging from characters motivation, Braddock's setting and the cast chemistry.
"Out of the Furnace" is currently released in theaters in New York and Los Angeles. It will be expanded nationwide tomorrow.
Read the full transcript of the press conference below:
There are amazing performances which are incomparable. I think you've outdone yourselves, especially you, Woody. What's important in this film is not only the family values, but the importance of the generational family work ethic. By picking a location in steel country like Braddock, Pennsylvania, how did you go about picking that location for the script and setting? For the actors, how did it inform your performances being steeped in that history of our country?
Scott Cooper: Well, I grew up in a small town along the same Appalachian Mountains range. As a grandson of a coal miner, I have grown up with these people. I've spent a lot of time in small town America. While I was touring with my first film, "Crazy Heart," I was reading a great deal about Braddock, Pennsylvania, or BRAD-DOCK as they say in Braddock.
What the town has undergone in the past five to seven years during the economic turmoil and loss of industry had really touched me. It's really important to me really shine to life of small town America, but what we Americans have undergone in these past five years. That blue collar life is something that I understood and resonated with me. I thought it was very underrepresented in American cinema. It was very relevant during the 1970s, which very much influenced this movie and "Crazy Heart."
I wanted to see that represented on screen again. I knew these people very well with their morays and values. It was very important to weave all of those things into a narrative in a personal way.
Christian Bale: It helps so much being on location, if you understand what I mean. It's like the difference between performing for the rectangle of the camera versus a world being created and then the camera finds things within that. There's a huge difference in that, because what it takes away is performance. You don't feel like performing. You're just kind of doing it. You're existing.
Casey Affleck: I think he summed it up pretty well. Especially in a place like where we were. There's a real story just in the way that it looks to see a place that was once one thing and is now something else. It has a lot of atmosphere. If there's a lighting set-up that takes 20 minutes, you can go into another room and you're not just staring at the back of a bunch of plywood. You're actually in another room in your own house where you're supposed to be, and it helps to ground you and keep you.
Woody, would you like to comment on that too?
Woody Harrelson: I'd like to say ditto. [Laughter]
Zoe Saldana: You walk in with this fear, wanting to see something that you can imagine being so heavy, and what you learn and you take from it is the strength that you're able to absorb from these people. It's very easy to leave when things go wrong, but to stick around and to basically give life to a town, because of everything that it gave you generation after generation after generation. That to me is what defines a true American. It's sticking together when it gets really rough. It is a town that has been hit very, very hard to an extent that I've been to places around the world that leave you with a big knot in your stomach and you feel like an elephant has just sat on your chest, and Braddock was definitely one of those cities. But once you sit down with the people, you kind of wish you had an ounce of the strength that they possess every single day by sticking around. That was something that I really was very moved by.
My question is to Woody, you were extremely intense, but at the same time you were cool and calm, which is a rare combination. Scary more than that. Did that come naturally to you or is that a technique that you've developed over the years?
Woody Harrelson: I think it's important when you're acting to be as relaxed as possible even if you're doing something intense. You're basically in a state of dynamic relaxation. I think that these other actors might agree with me. Did that answer your question?
Is that natural for you or did you have to develop that?
Woody Harrelson: I didn't feel there was anything natural about playing Harlan DeGroat. (to Scott Cooper) What was it you told me when we finished?
Scott Cooper: The very last shot of the film was the very first scene that we shot at the drive-in. When we wrapped, Woody walked over to me and he hugged me and he said, "I have never wanted to shed a character so badly in my life." [Laughter] And truly, for me, I wanted Woody's character to represent the very worst of America and Christian's character to represent the very best of America. I wanted that kind of dichotomy and hopefully we succeeded.
I just want to quickly say that as someone who's had a very unremarkable career as an actor, if you feel like you have a little bit of talent as an actor, that once you see these four actors, and you see their work, and you see it on the other side of a lens, that you quickly realize there's a difference between being very modestly talented and gifted as they are, and it's a real treat to direct these actors, I have to say.
Christian Bale: But that being said, you need the right environment and he still creates that environment. It doesn't matter.
Zoe Saldana: That's true.
Zoe, your character is torn between these two guys. How did you wrap your head around being involved and loving both of these men? The scene on the bridge was really amazing.
Zoe Saldana: I think Lena has been torn by her life. She's probably had a rough life. I needed to build that for her and to understand that. I had endless conversations with Scott and we came to that conclusion that she hasn't had it easy. I needed to know why she just couldn't stick by the person that she truly, truly loved and she went with somebody that worked in law that symbolically is going to keep her safe. It has to do with her inability to cope with danger and pain. And so, I think that being torn between two men that have been really good to her is small potatoes in comparison to the torture that she has to live with herself knowing that she just has to make decisions that are going to protect her physically.
For Mr. Cooper, with regard Christian Bale's character, I thought it was interesting how Bale's character in a couple of moments he was shown in church or a chapel. You often don't see something casually [since] movies tend to shove [religion] down your throat. I thought it was interesting that this guy is going through some dark things. Was there any particular backstory? What made you have the character to be that way?
Scott Cooper: Well indeed, this is a man who as I was writing this character, I always thought of him as a very good man who is beset on all sides by a relentless fate. It was based on someone in my life who has suffered a great deal of tragedy and pain and loss, and who is one of the most positive people that I know, and is someone who has given me a great source of inspiration. That particular man's faith has carried him through, whether he's asking for absolution or for redemption or whatever it is that he's asking for in those very quiet and personal moments. In these small communities throughout America, and I'm certain around the world, people all pray to different gods and they all look for different things when they go to houses of worship and spirituality. It was important for me to have Russell Baze ask for that type of spirituality and that faith as he's certain that he's doing things that are very morally questionable and things that have happened in his life that through twists of fate and circumstance have put him in the position he's in. Thank you.
Scott, you talked earlier about what appealed to you about telling this story. I wondered if you could elaborate a little bit on what it was like to have the opportunity to bring it to life on the screen with such a talented group of actors?
Scott Cooper: Directors go their whole career without being able to tell personal stories and to work with a cast as talented as they are. I don't even consider it work. I honestly and sincerely consider it a privilege to see the type of work and the preparation and the care that these actors put into helping me realize my vision. There was no ego on the set. They were always questioning. They were taking a script that was decently written and elevating it in every way, and making me a better filmmaker, and making me really understand more about who I am as a person. After the modest success of my first film, I found it very daunting to have to live with that kind of burden of expectations. For someone who grew up with very little money and who had very little money after "Crazy Heart," you can get tempted to make movies for the wrong reasons. And when you have two little girls who want you to make that movie, or need you to make that movie, you just can't. You have to be very true to yourself and to your artistic world view. I chose to tell a personal story. When you tell a movie like this that's as emotionally charged as this is—it's a risk. Certainly, I could have taken a much less risky path after the success of my first film, but as one of my great cinematic heroes, Francis Coppola, would say, "If you aren't taking the highest, greatest risk, then why are you a filmmaker?"
Casey, as the veteran returning home from Iraq, I thought you gave a very raw, real performance particularly in the scene where you're describing all the things that you had seen over there. Was there any special preparation that you did for that role?
Casey Affleck: Well, not anything special, but I did some preparation. That preparation starts just with reading the script over and over and trying to absorb it and then talking to Scott a lot about where he's coming from because it's not really a part of the movie. It doesn't delve into too much which is good. And then, watching some documentaries and things like that. And then, talking to some veterans and just trying to piece together as much as you can what that experience might be like for somebody and for a lot of these guys. These guys now, in these wars, have done more tours than the average soldier in other wars just because it's not a volunteer army and they're careerists. They spend a lot of time over there with a constant level of anxiety and understanding what those symptoms are when they come back with some post-traumatic stress disorder. There's the depression, the frustration, the alienation and feeling like people don't want to hear what their experiences were and how lonely that could feel. And then, you have to forget about all that stuff and just be in the living room. Then there you are talking about cleaning the bedpan or something.
You just hope that all that stuff imbues whatever moment you're in and not try to bring it to every little scene. It's not to carry around your history in that way. It's just sort of background noise and you hope that whatever research you've done bubbles up to the surface at the right time when you're playing a scene that suddenly that it's about beer and then suddenly you're sharing some experience that you didn't were planning on sharing. You've done the work and so you just hope at the right moment it clicks and makes sense.
For Casey and Christian, your scenes are amazing and it genuinely feels like there is a family relationship between you. Did you know each other before this? Did you do anything before the movie started to establish that [chemistry]?
Christian Bale: Not really. It just happened to me. I didn't even know what had been going on. I'd been saying, "We want Casey for the role." There were a lot of conversations about that, and Scott and I just kept saying it, and eventually we were saying, "We're not doing it without Casey." I didn't actually meet Casey until we were doing that little camera test in Pittsburg just a couple of days before we started. (to Scott) All of the prison stuff was done in two days?
Scott Cooper: The very first two days.
Christian Bale: The very first two days of filming, we did all that prison sequence stuff. We had just gotten thrown into the deep end which was nice. No. That was it. It just happened. Casey's a fucking great actor and he was wonderful to work off of and it just came good.
What about you, Casey?
Casey Affleck: We spent weeks and weeks together. [Laughter]
Christian Bale: I forgot all of that. I was just lying. It blasted off my head. No. I did. Sorry mate, I forgot that.
Casey Affleck: No. It was like you said, but I don't know, sometimes it works. It's awkward to say because he's sitting right there, but I think he just makes everyone better around him and is an anchor of reality. If someone's in a scene with you and they're listening to what you say and they're looking at you in that way, then you're having a real conversation and they are real feelings and relationships. The whole thing feels a little bit more real in some way. I would have to attribute whatever apparent chemistry or relationship there is to that. And those first couple day in the prison, those were hard for me. It's always hard to get right into something. I'm usually terrible the first week. On the very first day or two, we were doing those scenes and Christian was very patient. I did and said some things that just immediately made me trust him and then it just went smoothly from there.
Scott Cooper: What you see between these two actors isn't something that you can learn in the Lee Strasbourg Institute or at Stella Adler. That is these two actors doing a great deal of investigative text work before. They won't say that, but they're also as talented as any two actors of my generation, simply put, and that's not the type of thing that you as a director can really manufacture. They really are two actors that are at the very height of their skill level quite frankly.
I want to ask about the physicality in this movie. This is not a movie that has hero punches. Everything is very realistic and therefore a lot scarier. I'm wondering for the actors, is there an added level of trust that has to be there between you so that Woody could be as scary as he needed to be but Casey still felt safe like he was going to eat your face?
Woody Harrelson: I was worried about him eating my face. [Laughter] We were just talking about that before because I was saying that working with Casey is kind of like working with a wild animal. You really don't know. Is he going to bite you? Do I want to be petted? [Laughter] It was a really great experience. And Christian is one of the greatest actors who ever lived. There's a level of confidence in the actor you're working with that really helps a lot. It makes all the difference.
"Out of the Furnace" is currently in theaters in New York and Los Angeles. It will open nationwide tomorrow.


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If you own a used book store and would like to advertise it in this space, click on the link below
Annual Book SaleFriends of the Arapahoe Library District at Koelbel Public Library 5955 South Holly Street303-798-2444
Friends of Loveland Public Library at Thomas M. McKee 4-H Youth & Community Building at The RanchLarimer County Fairgrounds and Events Complex,5260 Arena Circle970-962-2712(I-25 and Crossroads Blvd., Exit 259)
Friends of the Danbury Libraryat Police Activities League (PAL) Building35 Hayestown Rd.Friends Voice Mail: 203-797-4568Friends E-Mail: email@example.com
The Mark Twain Library at Redding Community Center37 Lonetown Road (Route 107)203-938-2545JUST FOLLOW OUR GREEN DIRECTION SIGNS!
57th Annual Mark Twain Library Book Fair
The Granddaddy of Book Fairs - we are the oldest - and one of the largest - in New England. All proceeds benefit the Library.
Over 60,000 items organized in 75 categories including:
And our celebrated COLLECTOR'S CORNER, featuring rare finds, first editions, signed copies - priced to sell.
Credit Cards Accepted for Purchases Only
Scanners and other wireless devices welcome in sales areas; stockpiling and "clearing" not permitted.
Air-conditioned comfort, handicapped accessible, refreshments sold, FREE parking
Visit the Mark Twain Library website for more information
Friends of the Library Book House430 North Main St.352-375-1676www.folacld.org(Across from Sun Trust Bank)
Planned Parenthood of the Heartland at Iowa State Fairgrounds4-H Building 3000 East Grand Avenue515-234-0414
Friends of Boise Public Library 762 River Street208-384-4076(Across River Street from the Library)
BOOKSTOCK!Another Huge Bookstock Sale!Laurel Park Place37700 W. 6 Mile Road(1/4 mile east of I-275)248-645-7840 ext 365
1 Million+ Books!YMCA Book FairQueeny Park550 Weidman Rd314-353-4960
BIG FALL SALE!!Friends of Henderson County Library at 1940 Spartanburg Hwy (Route 176)(next door to Dollar General)
Brigham City Library26 East Forest St435-723-5850
Jenson Books70 E 400 N435-753-JENS (5367)
Logan Library255 North Main St.435-716-9123
North Branch Library475 East 2600 N801-337-2617(enter west side of bldg)
Friends of Park City Library 1255 Park Ave435-615-5600
AAUW - McLean, VA ChapterSpring Hill Recreation Center1239 Spring Hill RoadMcLean, VA 22102703-527-4206 (AAUW Contact)


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Same location for 40 years. Established clientele with great reputation. Prime corner lot location. Cozy neighborhood, authentic feel. Strong cash flow.
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Located in busy Camelback Road & 40th Street Area of Phoenix. Within apartment complex with 560 units and is centrally located across from main pool area. Featuring fresh deli sandwiches, beer, wine and household market goods
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A beautiful little trip to Mt Coot-tha. The sight was clear and stunning towards the top. A little walk around the botanical gardens afterwards which was fun as well as stumbling upon the most photogenic lizard ever.
Lovely day with a few snaps to compliment it.
This day was all about angles and trying to get more out of the photos. The lizard was definitely the highlight of the day as he just sat there and was lapping up the attention I was giving him.


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SAN JOSE MUSEUM OF ART'S Spectrum Gala - 45th AnniversarySeptember 13th, 2014
Sarah Ratchye, show more of Sarah Ratchye photos or correct name Peter Lipman, show more of Peter Lipman photos or correct name Susan Krane show more of Susan Krane photos or correct name
For a commercial or editorial license, please email email@example.com.
Perfect for printing with no watermark up to 8x12. If you need something larger, we can usually do a custom print.
Perfect for screen and social, but not great to print. There is a small watermark in the lower right corner (you can crop it out).


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Category: Themeforest | eCommerce
Tags: Themeforest | PSD Templates | nulled | free download | theme | script | rip | Moda | Opencart | theme
In archive: 2016 | April 2016 | 20.April 2016
The theme is compatible with Opencart 1.5.5.x/1.5.6.x/2.0.x/2.1.x and 2.2.x Valid HTML5Build My Ecommerce – Moda video review
It is very easy to install and customize. It ships with a powerfull customization panel that is active only if the admin user has permissions to modify the layout.
You can change colors, fonts and section visibility directly on your live website and save them directly without going to admin.Feature overviewOne page checkout module
Your store will have less abandoned carts andhappier customers with the integrated one page checkout module and buy now buttonOne click sample data import
Get an exact replica of the demo on your freshlyinstalled opencart with completemodule configuration and product images with one clickFaceted search
Allow users to find products faster with faceted search with price slider and multiple filters like categories, brands, product filters and product attributes.
* Currently available only for 0C 2.0.xLogin popup
Customers can quickly loginwith opencart or facebook/google accountsDrag and drop to cart
Customers can drag and drop products to the floating cart at the bottom of the pageCart popup
Add to cart notification with product image and direct checkout and cart linksSpeed cache module
Maximum performance with the fastest opencart page caching, the speed cache module bypases opencart code completley serving the page without even opening a database connectionHTML/CSS/JS speed optimization
CSS and JS concatenation and minification for fast page loading helping not only customers with a speedy navigation but also useful for SEO, loading speed has become a significant search engine ranking factor.Social connect
Allow customers to easily create an account with social media services like facebook/google etc with the integrated social authentication moduleResponsive and Mobile Ready
Expand your store on the mobile ecommerce marketwith full suport for mobiles and tables
Built on bootstrap 3 mobile first foundationLive visual panel editor
Change color schemes, fonts, colors and othersettings live on your store
All changes show live and then can be permanentlysaved with just a clickPowerfull products module
Custom products module suports multipledisplay types likefilter, tabs, carousel, tabs + carousel,grid and list
from multiple sources like autocomplete,latest, popular, also bought, category, bestsellers,recently viewed, manufacturer etcAnimated product filtering
Show products from multiple sourceswith animated filtering
Feature included in the custom products module for maximum flexibilityAdvanced Module Filter
With the filter option included in alltheme modules you can choose to display a moduleonly for a certain category/product/information pageImage hover swap
Show a different image on hover to display a productfrom a different angle, color etc
Quicly show users variations ordifferent features of the productAjax search autocomplete
Search box will show product suggestionsas soon as the user enters a few letters
Product image is shown along product title andthe user can directly click to go on product pageMegamenu module included
You can have powerfull menu configurations andinclude products and custom html as menu items
The module admin interface is very easy to use andhas en expandable drag and drop category treeCustom products tab module
Add custom tabs to product pages
With integrated module filter show certain tabs fora specific product only or for all productsGoogle rich snippets
Improve google search usability for your storewith google rich snippets
Have better SEO tools for your storePowerfull sequence slider module
Add both slider backgrounds and overlay imagesto your slides
Along with animated text andleft/right/center alignment optionsPowerfull sideblock module
Add sideblocks with anything from youtube or vimeovideos to social plugins like twitter or facebookand custom html
Choose any font awesome icon andcolor combination for the sideblockBlog support
The theme includes full support for the popular freepav blog opencart module
Use the extra blog features of your store formarketing or SEORTL and Internationalizaton
You can easily switch to RTL language from module panel
also with opencart wide support for language packs you can sell in any international market and have global exposurePowerfull banner module
The included banner module has many configuration options and the posibility to add both text, icons and images
Additional options like banners per row are possibleGoogle maps module
Add google maps module anywhere on your store with the option to add unlimited markers and text
Useful to add your store locationsQuick view
With quick product view, users can browse products faster without changing pagesCloud zoom
The theme has builtin cloud zoom plugin to allow image zooming without modals
Usable and popular way to display product imagesNo core modifications
The theme does not alter any opencart files during installation
This ensures that there are no conflicts with vqmods or extensions are possibleCatalog mode
If you just want to showcase your products and you don't need people to buy your products switch to catalog mode and disable prices, cart and checkout
You can at any time switch back and also start selling your products500+ Google fonts to choose from
You can set any font from the google fonts list to any element on your store from the live customization panel
Personalize your store with stylish and modern typography
Theme requires PHP 5.3.0+Admin modules
user: demo
password: demo
Theme FAQChange log
Source: themeforest.net
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