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Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown.
TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1.
Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.
The tail-anchored (TA) membrane proteins include diverse family members such as cytochrome b5, DMPK A, & C, Bcl-2, Tom, and Sec61 β & γ which are critical for cell function [1-8]. Several of these proteins, such as Synaptobrevin, are important in vesicle transport and membrane fusion [9]. The tail-anchor of TA proteins is defined as the C-terminal hydrophobic transmembrane domain which may be flanked by hydrophilic amino acid residues [9,10]. The Tail-Anchors can target proteins to a wide range of subcellular compartments including the ER (endoplasmic reticulum) [11-13], the MOM (mitochondrial outer membrane) [14-17], peroxisomes [18,19], the perinuclear membrane [20] and the chloroplast outer envelope in plants [21]. The molecular mechanism by which tail anchored proteins target specific membranes is of much interest. There appears to be no consensus amino acid sequence in the different TAs of proteins that dictate their targeting. In fact the sequence of the amino acids in the transmembrane domain has been shown to be irrelevant in synaptobrevin, where a poly-leucine tail (13 amino acids) targets the protein to the ER [22]. However, all tail anchors are predominantly hydrophobic in nature, as determined from the amino acid sequence of the membrane spanning region. Some studies indicate that there may be recognition motifs contained within the TA for proteins such as PEX26 and PEX15p, which cause them to target the peroxisome [23]. No such motifs have been identified for TAs which target proteins to the ER or the MOM. However, some TAs which target proteins to the MOM, are flanked by positively-charged residues adjacent to the membrane-spanning region which are believed to dictate their targeting to either the ER, or both the ER and the MOM [2,24-26]. This has led to the view that the p ositive charges individually or collectively in and around the TA determines its subcellular targeting [21,26]. Other studies by Biellharz et al., [20], Brambillasca et al., [27] and Wattenberg et al., [28] have provided evidence that the hydrophobicity of a tail anchor sequence itself can also influence which organelle it will target. Weather this is a unifying concept involved in targeting all TAs remains to be explored. The Sarcolemmal Membrane Associated Protein (SLMAP, formerly known as SLAP) is a tail-anchored protein which can carry two alternatively spliced TAs [8,29]. All SLMAP isoforms are encoded by one gene and many SLMAP isoforms have now been shown to be expressed in a tissue specific manner with proposed roles in myoblast fusion, excitation-contraction coupling and centrosomal organization [8,29-32]. The largest SLMAP isoform, the 91 kDa SLMAP3, is ubiquitously expressed in all tissues whilst the smallest isoform, the 34 kDa SLMAP1, is only expressed in cardiac and muscle tissue [30]. Each SLMAP isoform shares the C-terminal region of the protein but the smaller isoforms do not possess the N-terminal structures such as the FHA domain [8].
We have previously shown that when TA1 or TA2 are encoded as part of SLMAP1 they direct it to subcellular membrane structures [31] but which organelles they target and how, has not been defined. In addition, we have found that SLMAP carrying either TA1 or TA2 can be differentially expressed in the same tissue [8] Further more, immunohistological and biochemical analysis implies that SLMAP localizes in different subcellular compartments within the cardiomyocytes including the sarcolemma, SR/ER, and the transverse tubules [29,31]. TA1 comprises 27 amino acid residues with a predicted transmembrane helix of 18 residues and TA2 comprises 30 amino acid residues with a predicted transmembrane helix of 19 residues [8]. In this study we identify how TA1 and TA2 affect the subcellular targeting of SLMAP1 in Cos7 cells and provide evidence which supports the view that the overall hydrophobic profile of a tail anchor is critical for determining its subcellular localization. Further more, our analysis indicates that SLMAP1 itself is not involved in vesicle transport but can target the ER as well as the mitochondria, possibly the MOM, where it may play previously unrecognized roles in the subcellular function of these organelles.
SLMAP is a component of intracellular membranes in non-muscle cells
The SR is derived from smooth endoplasmic reticulum, an extensive membrane-bound organelle, which contributes to general intracellular calcium regulation in eukaryotic cells [33]. In view of the abundant SLMAP expression in the SR of cardiac cells and the ubiquitous distribution of the 91 kDa SLMAP varient [30], we sought to determine whether this SLMAP isoform is also a component of membrane systems (ER, Golgi) in non-muscle cells. Microsomes were prepared from the postnuclear supernatant of rat liver homogenates and further separated into ER and Golgi fractions by sucrose gradient centrifugation (Figure 1) [34]. Protein fractions from each gradient were collected and separated by SDS-PAGE and subjected to immunoblot analysis using anti-SLMAP antibodies as well as specific markers of the ER (calnexin) and a cis-Golgi marker (α-mannosidase). A 91 kDa SLMAP isoform, consistent with the molecular size of the full length SLMAP protein, distributed with calnexin in fractions B1 and SGF3 but was not enriched in the fractions with the highest levels of calnexin (S3, C1 and C2) (Figure 1). SLMAP was absent in the stack Golgi fraction where α-mannosidase was enriched, and was present in SGF3 and SGFL together with some calnexin (Figure 1). These data suggest that SLMAP is not a resident of the Golgi although it may be present in specific regions of the ER in the liver. It seems likely that the difference between the distribution of SLMAP and calnexin is due to a combination of the properties of the tail anchors in the liver SLMAP as well as the lipid composition in the different forms and regions of the ER.
Figure 1. Sub cellular distribution of SLMAP from liver. Rat liver homogenate was fractionated by sucrose gradient centrifugation. Western blot analysis demonstrated that the cis-Golgi marker, α-mannosidase resides in the stacked Golgi fraction 1 (lane SGF1). Endogenous SLMAP (91 kDa) proteins are distributed among fractions where the ER marker calnexin is expressed (lanes B1; S3; SGF3). H (homogenate); PNS (postnuclear supernatant); SGF (stacked Golgi fraction); P (pellet).
Tail anchors in SLMAP target distinct subcellular membranes
In order to determine weather the two carboxyl-terminal TAs utilized by SLMAP can target to distinct membrane structures, 6Myc-tagged SLMAP1 carrying TA1 or TA2 was transfected in COS7 and its localization monitored relative to various components of intracellular organelles. SLMAP1 is a 34 kDa isoform which is expressed predominantly in cardiac and muscle tissue, and was found to be an integral membrane protein [30]. Co-staining with the ER marker calnexin demonstrated significant co-distribution with 6Myc-SLMAP1-TA1 at reticular formations (Figure 2A; a–c); however limited co-distribution was observed in cells expressing 6Myc-SLMAP1-TA2 (Figure 2A; d–f). Since SLMAP contains a coiled-coil leucine zipper involved in homodimerization [32], we investigated whether this motif will influence targeting. As shown, (Figure 2A; g–i) a deletion mutant lacking the leucine zipper (ΔLZ) 6Myc-SLMAP1-TA2ΔLZ had identical targeting when compared to the undeleted, expressed protein. Anti-ERGIC-53 monoclonal antibodies were used to detect the ER-Golgi intermediate compartment and consistently labelled fine punctuate structures concentrated at perinuclear sites in COS7 cells [35,36]. This distribution pattern was distinct from that of either 6Myc-SLMAP1-TA1 (Figure 2B; a–c) or 6 Myc-SLMAP1-TA2 (Figure 2B; d–f). Whereas Myc staining demonstrated that TA1 and TA2 direct SLMAP1 to perinuclear sites, no co-localization with the anti-Golgi58K monoclonal antibody was noted (Figure 2C; a–i). Further, the fungal metabolite Brefeldin A, a widely used agent that disrupts the structure of the Golgi apparatus [37,38] did not alter the subcellular distribution of SLMAP1 (data not shown).
Figure 2. Subcellular membrane distribution of SLMAP. (A) COS7 cells transiently transfected with 6Myc-tagged SLMAP variants encompassing either TA1 (A; a-c), TA2 (A; d-f) or ΔLZ_TA2 (g-i) were stained with anti-calnexin polyclonal antibodies (b, e, h). The overlay of the anti-Myc and calnexin in these cells is shown in A; c, f, i. SLMAP distribution with respect to the secretory system. (B) COS7 cells transfected with either 6Myc-SLMAP-TA1 (B; a-c) or 6Myc-SLMAP1-TA2 (d-f) were stained with anti-SLMAP (B; a, d) and anti-ERGIC-53 (B; b, e), with the overlay shown in B; c, f. SLMAP distribution with respect to the Golgi apparatus. (C) COS7 cells expressing either 6Myc-SLMAP-TA1 (C; a-c); 6Myc-SLMAP-TA2 (C; d-f) or 6Myc-SLMAPΔLZ-TA2 (g-i), were co-stained with anti-Golgi 58 K (C; b, e, h) and anti-SLMAP (a, d, g), with the overlay shown in C; c, f, i. Scale bar = 10 μm.
Disruption of the cytoskeleton and SLMAP localization
We examined the localization of endogenous SLMAP with anti-SLMAP antibodies and compared this with the ER marker calnexin using anti-calnexin antibodies and a clear co-distribution of these two proteins is evident (Figure 3A; a–c). The importance of cytoskeleton for the organization of the membrane organelles such as the ER has been demonstrated by the pharmacological disruption of microtubules [33]. In cells treated with the microtubule depolymerising agent nocodazole, the ER membrane collapses and aggregates around the nucleus [39-41]. Under conditions that disrupt microtubules, the distribution of the ectopically expressed ER-membrane associated SLMAP1 variant (6Myc-SLMAP1-TA1) was altered from a reticular-like distribution (Figure 3B; a) to an aggregate-like distribution (Figure 3B; b). These nocodazole-induced structures were not however observed in COS7 cells expressing SLMAP1 deletion mutant lacking the TA (6Myc-SLMAP1ΔTA) (Figure 3B; c). The effect of nocodazole on microtubule assembly was confirmed by staining with α-tubulin, which illustrated a distinct redistribution of the cytoplasmic microtubules (Figure 3B; d–f). These observations imply that SLMAP1-membrane associations require an intact microtubule network and would be consistent with the distribution of SLMAP1-TA1 at the ER membrane.
Figure 3. SLMAP localizes to the ER and its associations are microtubule dependent. (A), COS7 cells were stained with anti-SLMAP (A; a) and anti-calnexin (A; b) and merged image (A; c). (B), COS7 cells were transfected with 6Myc-SLMAP-TA1 (B; a, b, d, e) and 6Myc-SLMAPΔTM (B; c, f) and stained with Anti-SLMAP (B; a-c) and anti-α-tubulin (B; d-f). Cells treated with nocodazole (B; b, c, e, f). (C) COS7 cells transfected with either 6Myc-SLMAP-TA1 (C; a, b, e, f) or 6Myc-SLMAP-TA2 (C; c, d, g, h) and stained with phalloidin (C; e-h) and anti-Myc (a-d). Treatment with cytochalasin D (C; f, h) did not alter the distribution of the 6Myc-SLMAP-TA1 (C; b) or 6Myc-SLMAP-TA2 (C; d). Scale bar = 10 μm.
To determine whether SLMAPs are associated with the actin cytoskeleton, COS7 cells expressing 6Myc-SLMAP1 fusion proteins were treated with cytochalasin D, an actin myofilament disrupting agent [42]. Depolymerization of the actin filaments with cytochalasin D did not alter the localization of 6Myc-SLMAP1 fusion proteins carrying either TA1 (Figure 3C; a, b) or TA2 (Figure 3C; c, d), whilst inducing actin-containing microfilaments to change from filamentous (Figure 3C; e, g) to punctuate structures (Figure 3C; f, h).
SLMAP and ER-Golgi transport
Comparisons of the predicted primary structure of SLMAP indicates general similarities with coiled-coil TA anchored proteins involved in transport of vesicles from the ER and the docking of vesicles at the Golgi [43,44]. Analysis of the amino acid sequences encoding SLMAPs (Accession No. AAA65597) also indicated the presence of a putative di-acidic sorting signal upstream of the carboxyl-terminal transmembrane domain. (EQE) Di-acidic motifs, defined by Asp/Glu residues separated by a variable residue (D/E)X(D/E), are often located in close proximity to tyrosine-based sorting motifs (YXXö) and are thought to mediate efficient export of the protein from the ER by interacting with the vesicular transport machinery [45,46]. In SLMAP a tyrosine-based sorting motif (YEKT) is present upstream of the di-acidic sorting motif. In view of these features we investigated whether SLMAPs are involved in vesicular transport from the ER to the Golgi. Previous studies have shown that overexpression of coiled-coil membrane proteins involved in vesicle trafficking from the ER to Golgi inhibit protein export from the ER [47]. To examine this possibility, COS7 cells were co-transfected with an ER-localized 6Myc-SLMAP1 and the GFP-tagged glycoprotein of the temperature sensitive strain of vesicular stomatitis virus (ts045-VSV-G-GFP), which serves as a marker of ER to Golgi transport. When maintained at the restrictive temperature (39.5°C), the viral glycoprotein could not be exported from the ER due to a thermoreversible-folding defect (Figure 4d) and consequently co-distributed with 6Myc-SLMAP1 (Figure 4a &4g). Proper folding and subsequent transport out of the ER to the Golgi and cell periphery was achieved when the ts045-VSV-G transfected cells were incubated at the permissive temperature (32°C). Whereas the GFP-labelled viral glycoprotein was transported out of the ER to the Golgi and plasma membrane under conditions that promote vesicular transport (Figure 4e), the 6Myc-SLMAP fusion protein did not appear to exit the ER (Figure 4b) as clearly indicated in the overlay (Figure 4h). Expression of the SLMAP1 lacking either the transmembrane domain or the leucine zipper motifs did not affect the ability of the viral glycoprotein to exit the ER at the permissive temperature (data not shown). COS7 cells co-expressing ts045-VSV-G-GFP and 6Myc-SLMAP1 were also incubated at reduced temperatures to monitor whether SLMAP1 cycles from the ER to the ERGIC [48]. At 15°C the membrane associated SLMAP1 was retained in the ER (Figure 4c), whereas the GFP-labelled viral glycoprotein was visualized as vesicular clusters concentrated at perinuclear sites (Figure 4f) consistent with localization at the ERGIC (Figure 4i). Collectively, these studies indicate that SLMAP1 is not exported via the vesicular transport process nor does elevated expression of SLMAP1 affect transport and is consistent with its absence from the stack Golgi fraction.
Figure 4. Overexpression of SLMAP does not affect VSV-G transport from the ER. COS7 cells were co-transfected with 6Myc-SLMAP and ts045-VSV-G-GFP. When maintained at 40°C (a, d, g), the GFP-tagged viral glycoprotein was retained in the ER (d) and co-distributed with the Myc-labelled SLMAP protein (a). At 32°C, ts045-VSV-G-GFP exited the ER and was observed at perinuclear structures as well as at the cell periphery (e); whereas the 6Myc-SLMAP (b) appeared to remain in the ER. In cells incubated at reduced temperatures (15°C), the GFP-tagged viral glycoprotein is redistributed to perinuclear punctuate-like structures indicative of the ERGIC (f). The localization of the 6Myc-SLMAP (c) remained unaltered in these cells. Overlay of the anti-Myc and GFP signals is shown in g, h, and i.
Hydrophobic profiles of the TAs in SLMAP
A single gene encodes SLMAP which undergoes alternative splicing to generate distinct isoforms with unique tail anchors which serve roles in myoblast fusion, centrosomal function and excitation-contraction coupling [29,31,32]. Exon XXIII (of SLMAP) encodes 27 amino acids which comprise the alternatively spliced TA, referred to as TA1. Exon XXIV encodes 30 amino acids which form the constitutively expressed TA, referred to as TA2 [8,29]. To more clearly define SLMAP targeting, the nature of the TAs in SLMAP was explored using the HMMTOP program which predicted that SLMAP's two alternatively spliced C-terminal exons contain a single putative transmembrane domain [49,50] (Figure 5A). TA1, encoded by Exon XXIII, contains a predicted transmembrane helix of 18 amino acids flanked by five amino acid residues at the N-terminal end and four residues at the C-terminal end. TA2, encoded by Exon XXVI, is predicted to contain a transmembrane helix of nineteen amino acids flanked by seven residues at the N-terminal end and five residues at the C-terminus. The two TAs have no sequence homology, but have similar properties, in that they are largely hydrophobic domains (Figure 5B). The major difference between the two TAs is the presence of a basic amino acid residue on either flank of the membrane spanning region in TA2, which are not present in TA1 (Figure 5A).
Figure 5. Primary sequence and hydrophobicity of TAs in SLMAP. Both of the alternatively spliced SLMAP tail anchors are predicted to encode a transmembrane helix according to the HMMTOP program. The transmembrane regions are shown in bold and underlined, the positively charged residues are shown in a larger font and bold for both TA1 and TA2 (A). The hydrophobicity of the TA1 (pink square) and TA2 (blue diamond) was calculated using the Eisenberg normalised scale, with a window size of 9, the relative weight for window edges was 100 (B).
Numerous reports have shown that the presence of positively charged residues in the regions flanking the membrane spanning region can cause a TA to target the MOM rather that the ER [2,24,25]. A simple examination of the flanking sequences of TA1 and TA2 shows that only TA2 possesses positively charged residues in this region (Figure 5A). This suggests that TA2 may target the MOM whilst TA1 could target the ER. However, some ER targeting TAs possesses positively charged residues in the flanking regions [2,3,14,22], so we might conclude that there may be additional factors involved in the targeting of TAs. It has been shown that the hydrophobicity of the transmembrane region can affect the membrane targeting of a TA [20,27].
The hydrophobicity of the TA1 and TA2 was calculated using the Eisenberg normalised scale with a window size of 9, the relative weight for window edges was 100% (Figure 5B) [51]. The hydrophobicity scores for the two putative trans-membrane domains differ in several regions. TA1 is more hydrophobic over the first five residues which form the N-terminal flanking region and the last five residues, which form the C-terminal flanking region (Figure 5B). However, the largest difference between the SLMAP TAs can be seen in the transmembrane region. TA1 is much more hydrophobic than TA2 between residues 10 and 20, with TA1 having a maximum score of 1.03 whilst TA2 reaches 0.8. This data led us to believe that the difference in hydrophobicity between the two TAs may be enough to result in differential targeting.
TA2 sequences can target SLMAP1 to mitochondria
The endogenous localisation of SLMAP in COS7 cells was examined using anti-SLMAP antibodies (Figure 6a) and compared with the mitochondrial marker TOM20 [52] (Figure 6b). The co-distribution (Figure 6c) shows that a significant amount of the endogenous SLMAP co-distributes with TOM20 however there are regions in the cell, where SLMAP is present without TOM-20. These regions would be consistent with the presence of SLMAP at the ER as noted by anti-calnexin staining (Figure 3A; a–c). The subcellular localization of the ectopically expressed SLMAP1 variants encoding TA1 and TA2 were further analysed with respect to Tom20 and MOM localization. Dual immunostaining using anti-Tom20 and anti-Myc antibodies showed that the 6Myc-SLMAP1-TA1 variant did not localize at the mitochondria (Figure 6d–e). A mitochondrion is indicated by the arrow in Figure 6e. Whereas, co-distribution of Tom20 with the 6Myc-SLMAP1-TA2 indicated that this variant may target the MOM, whilst still being present in a filamentous structure throughout the cytoplasm in a similar way to the TA1 construct (Figure 6g–i). The filamentous structure targeted by 6Myc-SLMAP1-TA2 is indicated by an arrow in Figure 6i. It seems clear that SLMAP1-TA2 targets the mitochondria but in addition it also shows co-localization with calnexin at the ER (Figure 2A; d–f) which is distinct from the exclusive localization of SLMAP1-TA1 at the ER (Figure 2A; a–c).
Figure 6. Endogenous SLMAP localises at the mitochondria and is targeted by the TA2. Cells were stained with anti-SLMAP (a) and anti-TOM20 (b) to analyze endogenous co-distribution (c). Cos7 cells were transfected with 6Myc-SLMAP1-TA1, and co-stained with anti-Tom20 (d) and anti-6Myc (e), the images were then merged (f) arrow points to an anti-Tom20 stained mitochondria. Cos7 cells transfected with 6Myc-SLMAP1-TA2 and co-stained with anti-Tom20 (g) and anti-6Myc (h) and the images were then merged (i) arrow points to anti-6Myc stained structure with no mitochondria present.
In order to determine the molecular properties of TA2 which are responsible for mitochondrial membrane targeting we performed mutational analysis of the tail anchor and flanking regions. We used site-directed mutagenesis to substitute hydrophobic residues for the positively charged residues in the flanking regions to see if TA2 was targeted to the mitochondria via these positively charged residues. The first amino acid in TA2, a lysine residue, was substituted with a leucine residue to remove the N-terminal flanking positive charge, to create the mutant TA2-K1/L (Figure 7A). The 27th amino acid, an arginine residue, was substituted with a leucine residue to remove the C-terminal flanking positive charge in the mutant TA2-R27/L (Figure 7B). These two mutations were combined in the mutant TA2-K1/L+R27/L to remove both flanking positively charged residues (Figure 7C). The Myc tagged mutant constructs, SLMAP1-TA2-K1/L, SLMAP1-TA2-R27/L and SLMAP1-TA2-K1/L+R27/L were transfected into Cos7 cells and compared with GFP tagged SLMAP1-TA2 for targeting properties. No change in the targeting of SLMAP1-TA2 was observed due to these mutations suggesting that the positive residues are not responsible for the targeting of TA2 to the mitochondria (Figure 8).
Figure 7. Primary sequence and membrane helix prediction of the TA2 mutants. Amino acid substitutions are indicated by red bold letters in a larger case. The amino acid residues are shown using the single letter code and the transmembrane region in each tail anchor is shown in bold and underlined. The sequences shown are from Mus musculus.
Figure 8. Targeting of SLMAP1-TA2 and charge. Wild type GFP-tagged SLMAP-TA2 was co-transfected into Cos7 cells with wild type Myc-tagged SLMAP-TA2 and co-stained with anti-GFP (a) and anti-6Myc antibodies (b) the overlay of the two shows that there is nearly complete overlap with a stronger GFP signal near the nucleus (c). Wild type GFP-tagged SLMAP-TA2 was co-transfected with Myc-tagged SLMAP1-TA2 K1/L and co-stained with anti-GFP (d) anti-6Myc (e) the overlay is essentially the same as the control (f). Wild type GFP-tagged SLMAP-TA2 was co-transfected with Myc-tagged SLMAP1-TA2 R27/L and co-stained with anti-GFP (g) and anti-6Myc antibodies (h) the overlay is essentially the same as the control (i). Wild type GFP-tagged SLMAP-TA2 was co-transfected with Myc-tagged SLMAP-TA2 K1/L + R27/L and co-stained with anti-GFP (j) and anti-6Myc (k) antibodies the overlay is essentially the same as the control (l).
The mutations introduced in TA2 above increased the hydrophobicity of the flanking sequences, without affecting the hydrophobic profile of the transmembrane region itself (Figure 9A). To test whether the hydrophobic properties of the transmembrane sequence in TA2 were important for the targeting of SLMAP1 to the mitochondria we constructed mutants with increased hydrophobicity in their transmembrane domain. Four mutations were made in TA2 to construct a mutant TA2 transmembrane region with similar hydrophobic properties to TA1 (Figure 9B). The alanine residues 10, 11 and 17 along with threonine 16 were all substituted with leucine residues to make SLMAP1-TA2 A10/L+A11/L+T16/L+A17/L (SLMAP1-TA2-4L) (Figure 7E). We transfected Myc tagged SLMAP1-TA2-4L into Cos7 cells and co-transfected with wild type GFP tagged SLMAP1-TA2. The TA2-4L mutant did not overlap with the wild type TA2 in many areas in the cell (Figure 10a–c). When we transfected Cos7 cells with the Myc tagged SLMAP1-TA2-4L mutant and co-stained with anti-TOM20 antibodies we saw no overlap with the mitochondrial marker (Figure 10d–f). We co-transfected the Myc tagged SLMAP1-TA2-4L mutant with a GFP tagged wild type SLMAP1-TA1 the observed overlap indicated that both constructs target SLMAP1 to the same cellular compartment (Figure 10g–i). Thus it appears that the TA2-4L mutant shares the targeting properties of wild type TA1.
Figure 9. Hydrophobicity of TA2 mutants. The hydrophobicity of the TA2-4L (light blue circle) and TA2-2L (green triangle) mutants was calculated using the Eisenberg normalised scale, with a window size of 9, the relative weight for window edges was 100, compared with wild type TA1 (pink square) and TA2 (blue diamond) (A). The hydrophobicity of the TA2-K1/L+R27/L mutant (brown triangle) was calculated using the Eisenberg normalised scale, with a window size of 9, the relative weight for window edges was 100, compared with wild type TA1 (pink square) and TA2 (blue diamond) (B).
Figure 10. Targeting of the SLMAP-TA2-4L mutant. The targeting of SLAMP1-TA2 in Cos7 cells can be altered to that of SLMAP1-TA1 by increasing the hydrophobic moment of the trans-membrane helix with 4 substitutions: A10/L, A11/L, T16/L, and A17/L. Wild type GFP-tagged SLMAP1-TA2 was co-transfected with Myc-tagged SLMAP1 TA2 with the 4 substitutions (Myc-TA2-4L) and co-stained with anti-GFP (a) and anti-6Myc antibodies (b) the overlay of the two shows that there is incomplete overlap (c). In Cos7 cells stained with antibodies to TOM20 (d) and transfected with Myc-TA2-4L and stained with anti-6Myc antibodies (e) there are large numbers of mitochondria which do not colocalise with any of the mutant SLMAP (f). Wild type GFP-tagged SLMAP1-TA1 was co-transfected with Myc-TA2-4L and co-stained with anti-GFP (g) and anti-6Myc antibodies (h) when the two images are overlaid there is almost total co-localization (i).
To determine whether a smaller change in hydrophobicity could produce a similar alteration of targeting, we constructed a mutant with two changes in the trans-membrane domain (Figure 9B). The alanine amino acid residues at positions 10 and 11 were substituted with leucine to create SLMAP1-TA2 A10/L+A11/L (TA2-2L) (Figure 7D). When we co-transfected Myc tagged SLMAP1-TA2-2L with wild type GFP tagged SLMAP1-TA2 in Cos7 cells we observed that the TA2-2L mutant only partially co-localised with TA2 (Figure 11a–c). The Myc tagged SLMAP1-TA2-2L was transfected into Cos7 cells and co-stained with anti-Tom20 antibodies. There was no overlap between the TA2-2L mutant and the MOM marker Tom20 (Figure 11d–f). We co-transfected Cos7 cells with Myc tagged SLMAP1-TA2-2L and wild type GFP tagged SLMAP1-TA1 and observed nearly complete overlap of the two (Figure 11g–i). The results of the TA2-4L and TA2-2L were largely identical, indicating both mutants share the same targeting properties, which were observed to be the same as wild type TA1.
Figure 11. Targeting of the SLMAP1-TA2-2L mutant. The targeting of SLAMP1-TA2 in Cos7 cells can be altered to that of SLMAP1-TA1 by increasing the hydrophobic moment of the membrane spanning section with 2 substitutions: A10/L and A11/L. Wild type GFP-tagged SLMAP1-TA2 was co-transfected with Myc-tagged SLMAP1-TA2 with the 2 substitutions (Myc-TA2-2L) and co-stained with anti-GFP (a) and anti-6Myc antibodies (b) the overlay of the two shows that there is incomplete overlap (c). In cells stained with antibodies to TOM20 (d) and transfected with Myc-TA2-2L and stained with anti-6Myc antibodies (e) there are large numbers of mitochondria which do not co-localize with any of the mutant (f). Wild type GFP-tagged SLMAP1-TA1 was co-transfected with Myc-TA2-2L and co-stained with anti-GFP (h) and anti-6Myc antibodies (i) when the two images were merged there was almost total co-localization (j).
The TA2-4L mutant has a transmembrane region that is only slightly less hydrophobic than the wild type TA1 (Figure 9B) They were found to share similar targeting properties despite the TA2-4L mutant still possessing positively charged amino acid residues in both of the regions flanking its trans-membrane domain, with the corresponding decrease in the hydrophobicity of these regions (compared with TA1) (Figure 7E). The TA2-2L mutant has a much less hydrophobic trans-membrane region than both TA1 and TA2-4L, but is still more hydrophobic than wild type TA2 over amino acids 6–15 (Figure 9B). However, it also shares the same targeting properties as TA1; despite it like TA2-4L possessing flanking positively charged amino acids (Figure 7D). It appears from this data that the TA2 transmembrane region is at the very edge of the hydrophobic range which will target a TA to the MOM. This may be the reason that TA2 is promiscuous in targeting SLMAP1 to mitochondria and ER.
The TA2-K1/L+R27/L mutant which has the positively charged amino acids replaced with leucine does have an increase in hydrophobicity compared with wild type TA2 but only in the regions flanking the transmembrane domain (Figure 9A). The hydrophobicity of the trans-membrane region of the TA2-K1/L+R27/L mutant remains the same as the wild type TA2 trans-membrane region (Figure 9A). These data suggest that the targeting of TA2 to the mitochondria is due to the reduced hydrophobicity of the transmembrane region in comparison with the TA1 transmembrane region rather than the presence of the positively charged amino acid residues in the flanking sequences.
Tail anchored (TA) membrane proteins are important in several cellular processes including neurotransmitter release, vesicle transport, membrane fusion and signal transduction [1-8]. All tail anchors comprise a C-terminal hydrophobic transmembrane region which may be flanked by positively charged residues which are promiscuous in terms of sub-cellular targeting [10]. These features are present in the SLMAP C-terminal region and may account for its distinct pattern of localization observed here in the liver versus Cos 7 cells compared with the cardiomyocytes [31]. It is notable that endogenous SLMAP co-localised with the ER marker calnexin and the mitochondrial marker TOM20 implying that it is like other TA proteins such as BCL2 which also reside in these distinct membranes [5]. The different TAs utilized by SLMAP appear to be responsible for the targeting of SLMAP1 to distinct subcellular structures including the ER and mitochondria, perhaps the MOM.
The information required for the targeting of SLMAP to intracellular membranes was not contained in specific amino acids that flank the predicted transmembrane domain but was dictated by the overall hydrophobic profile of the TA. We found that SLMAP1 could target either the ER alone or the ER and the mitochondria in Cos7 cells depending on which of the two alternative tail anchors (TA1 or TA2) were being expressed. The disruption of the cytoskeleton with nocodazole showed that the distribution patterns of SLMAP1 are dependent on an intact microtubule network. This is wholly consistent with changes in distribution noted for other ER resident proteins, and the known role of the cytoskeleton in anchoring intracellular membranes in place [39,53]. It is interesting to speculate that SLMAP may act as a potential molecular link between intracellular membranes and the microtubule based cytoskeleton. Several membrane associated proteins provide a link between intracellular membranes and the microtubule cytoskeleton including CLIPs [53] and the ER membrane protein p63 [39,54]. Such interactions, which may also involve SLMAP, are crucial for the positioning and structural maintenance of subcellular organelles [55].
Tail anchors comprise a single membrane spanning helix of 17 to 21 amino acid residues at the C-terminal end of the protein [9,10]. Investigations have shown that the replacement of basic residues in the regions flanking the membrane helix with hydrophobic residues can alter the targeting of TAs [2,24,25]. It would appear that the presence of positively charged residues in the flanking regions increases the likelihood of the TA targeting the MOM instead of the ER [26].
The two different tail anchors in SLMAP are generated by alternative splicing and both appear to target the protein to subcellular locations. Both TA1 and TA2 target SLMAP1 to the ER. TA2 also targets SLMAP1 the mitochondria as evidenced by co-localization with Tom20. We found that this differential targeting was due to the lower hydrophobic content of the trans-membrane helix in TA2 when compared to the alternatively spliced TA1. Immunohistochemistry indicates that TA1 directs SLMAP1 predominantly to the ER and not the mitochondria probably due to the more hydrophobic nature of the transmembrane domain. We were able to prevent TA2 from targeting the mitochondria by the substitution of moderately hydrophobic amino acids with the highly hydrophobic leucine residues implying that the important targeting information in TA2 is the relative hydrophobicity of the transmembrane region. In this regard Bielharz et al., [20] observed a similar shift in the targeting of Fis1 from the MOM to the ER when the hydrophobicity of its TA was increased.
We found no evidence to suggest that the positively charged amino acids which flank the membrane spanning helix are responsible for the targeting of TA2 to the mitochondria per-se. This is in contrast to the view that basic amino acids flanking the TA are responsible for MOM targeting [2,7,14,24,25]. It seems more likely that in these cases the substitution of the positively charged, and therefore highly hydrophilic, amino acids results in an increase in the overall hydrophobicity of the TA and altered targeting. However, this did not occur in our TA2 K1/L R27/L mutants despite the hydrophobicity of the flanking regions being increased to a level above that of the TA1 isoform. Recently it has been shown that artificial TAs require a moderately hydrophobic transmembrane region to target the MOM [28]. Consistent with this speculation, the directed substitution of 2 amino acid residues in the flanking region in TA2 of SLMAP1 did not sufficiently change hydrophobic nature to shift targeting.
The presence of the two alternatively spliced exons encoding tail anchors which target SLMAP1 to different locations suggests SLMAP1 may perform multiple subcellular functions. Given the coiled-coil nature of the majority of the SLMAP protein, it may be that it tethers proteins into place on both the ER and perhaps the MOM. Alternatively, it may be involved in tethering of the two membrane systems together through homo/hetero-dimerisation. Differential levels of expression of SLMAP with the different TAs, suggest that SLMAP may serve varying subcellular roles in tissue specific manner [8,30,31]. In this regard, the expression levels of SLMAP were found to be important for normal myoblast fusion [29]. SLMAP was not found to be localized with the vesicle transport mechanism nor did its over expression affect vesicle transport in Cos7 cells, this implies that it is not involved in intracellular trafficking.
While the mechanism of TA protein membrane insertion remains to be defined, it appears that both the SRP (signal recognition particle) and a novel mechanism utilizing ATP appear to be involved [56,57]. Which of these mechanisms is utilised by SLMAP remains unknown.
Our data here supports the view that the sub-cellular targeting of SLMAP is determined by the overall hydrophobicity of its alternative tail-anchor (TA). Further, the basic amino acid residues in the regions flanking the transmembrane helix contribute to the overall hydrophobic profile of the TA and its targeting rather then the individual residues being a targeting signal. Further more, the potential association of SLMAP with the MOM implies additional roles for this molecule in mitochondrial function.
Membrane helix predictions were performed using the HMMTOP program [49,50]. Hydropathy calculations were performed using the ProtScale tool http://www.expasy.org/cgi-bin/protscale.pl webcite with the Eisenberg algorithm using a window size of 9 and the relative weight for the window edges was 100%. [51].
DNA constructs
Since the targeting of the tail anchored proteins resides at the C-terminal regions in general we used GFP and 6Myc-tagged expression constructs of the naturally occurring SLMAP1-TA1, SLMAP1-TA2, as previously described [29,31,32]. Site directed mutagenesis was performed using the Stratagene "Quikchange" kit. Template DNA concentration was 30 μg per reaction. The K1/I mutant (AAA/ATA) was created using 5' (GGA AAT AAT ATA CCC TGG CCC) and 3' (GGG CCA GGG TAT ATT ATT TCC). The R27/I mutant (AGA/ATA) was created using 5' (CCA GGT CTG GCC ATA GCT TCT CCG TG) and 3' (CA CGG AGA AGC TAT GGC CAG ACC TGG). The K1/I+R27/I mutant was created by performing SDM on the K1/I mutant using the R27/I primers (5' (CCA GGT CTG GCC ATA GCT TCT CCG TG) and 3' (CA CGG AGA AGC TAT GGC CAG ACC TGG)). The P4/L mutant (CCC/CTC) was created using 5' (ATA CCC TGG CTC TGG ATG CCC) and 3' (GGG CAT CCA GAG CCA GGG TAT). The A10/L + A11/L mutant (GCT/CTT + GCC/CTC) was created using 5' (ATG CCC ATG TTG CTT CTC CTG GTT GCG GTG) and 3' (CAC CGC AAC CAG GAG AAG CAA CAT GGG CAT). The T16/L + A17/L mutant (ACA/CTA + GCT/CTC) was created using 5' (CTG GTT GCG GTG CTA CTC ATC GTG CTG TAT) and 3' (ATA CAG CAC GAT GAG TAG CAC CGC AAC CAG). The A10/L+A11/L+T16/L+ A17/L mutant was created by SDM on the A10/L+A11/L construct using the T16/L + A17/L creation primers (5' (CTG GTT GCG GTG CTA CTC ATC GTG CTG TAT) and 3' (ATA CAG CAC GAT GAG TAG CAC CGC AAC CAG)).
Cell culture, transfections and cytoskeletal disruptions
COS7 African green monkey kidney and C2C12 cells were maintained at 37°C in Dulbecco modified essential media (DMEM) supplemented with 10% heat inactivated fetal bovine serum and antibiotics. Transient transfection experiments were performed using the fugene™ (Roche Biochemicals) transfection reagent according to the manufacturers' specifications. Disruptions of microtubules were induced in COS7 cells with 10 μM nocodazole (Sigma-Aldrich) for two hours at 37°C. Filamentous actin was disrupted in COS7 cells with 1 μM cytochalain D (Sigma-Aldrich) for 2 hours at 37°C. Cell Culture and Immunohistochemistry. Transfections were performed as previously described [30], and visualised using specific antibodies, anti-Myc 9E10 (Roche), anti-TOM20 (Santa Cruz), anti-GFP (Roche), and anti-Calnexin (StressGen) with an Axiophot (Carl Zeiss) fluorescent microscope and images captured as described previously [29]. For endogenous co-localisation studies either C2C12 cells or COS7 cells were stained with anti-SLMAP antibodies and either anti-Calnexin or anti-Tom20.
Subcellular fractionation of the Golgi and ER
Stacked Golgi fractions (SGF) and endoplasmic reticulum fractions were isolated from rabbit liver according to the method described by Taylor et al., [34]. All procedures were performed on ice in the presence of protease inhibitors (PMSF, leupeptin, aprotonin, pepstatin A). Freshly removed livers were minced and then homogenized (0.5 M phosphate buffered sucrose. 100 mM KH2PO4/K2HPO4 pH 6.5 and 5 mM MgCl2). The homogenate was centrifuged at 1500 × g for 10 minutes to remove unbroken cells, debris and nuclei. Postnuclear supernatant (PNS) was loaded onto a sucrose step gradient (1.3 M sucrose, 0.86 M sucrose, PNS in 0.5 M sucrose, 0.25 M sucrose) and centrifuged at 100,000 × g for one hour. Fractions collected from the gradient included S1 (0.25–0.5 M interface), A1 (0.5 M layer), S2 (0.5–0.86 M interface), B1 (0.86 M layer), S3 (0.86–1.3 M interface), C1 (1.3 M layer), and pellet (P). S2 fraction was adjusted to 1.15 M sucrose, and then overlaid with 1.0 M sucrose, 0.86 M sucrose and 0.25 M sucrose. This sucrose gradient was centrifuged at 76,000 × g for 3 hours. Fractions collected included: SGF1 (0.26–0.86 M interface), SGF2 (0.86–1.0 M interface), C2 (1.0 M layer), SGF3 (1.0–1.15 M interface), and SGFL (1.15 M layer). Protein content of each fraction was determined via the BCA protein assay (Pierce). Equal amounts (10 μg) of each fraction were loaded onto a 10% SDS polyacrylamide gel. ER-Golgi protein transport assays The ts045-VSVG-GFP expression plasmid was donated by Dr. Lippincott-Schwartz [37]. The plasmid was co-transfected with 6Myc-tagged SLMAP-pcDNA3 constructs into COS7 at 40°C for 15 hours. Cells were then shifted to either 32°C for one hour or 15°C for three hours to monitor the transport of ts045-VSVG-GFP from the ER to the ERGIC, Golgi and plasma membrane.
Authors' contributions
JB conducted the experiments on the site directed mutagenesis and the hydrophobicity predictions while RG carried out the trafficking and biochemical studies and MS analyzed sequences and made constructs. BST conceptualized the experiments and drafted the manuscript together with JB, RG and MS. All authors read and approved the final manuscript. JTB and RMG contributed equally to this work.
This work was supported by an operating grant from the Heart and Stroke Foundation of Ontario (#T5821) to BST.
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text: On Tue, 14 Feb 2012 17:28:34 -0800 (PST) Fernando de Oliveira <famobr at yahoo.com.br> wrote: > It took a while to answer you, because there were localization issues. Last and annoying one was the keyboard layout indicator on lxpanel which was displaying US, instead of BR as default. I was going to write this, and suddenly it switched to the right one without my intervention. > > Hoping to some env variables could be used when calling X, I have adapted a little your instructions: > > user=fernando > su - ${user} -l -c "env LANG=pt_BR.utf8 xinit ~/.xinitrc &> ~/.x-session-errors" That looks fine, but for what it's worth, I set environment variables like LANG in /etc/profile and then source /etc/profile from ~/.bash_profile. Now that I think back, I seem to recall that I first did this so that lxpanel would show the date correctly (it was showing it in the American style). I like lxpanel. It's not perfect but it's good enough and it only has 2 dependencies. > If you tell me that for some reason I should do /etc/X11/xinitrc instead of ~/.xinitrc, I will change immeadiately. It's fine, ~/.xinitrc is the default for xinit so you don't need to specify it. I just put the file somewhere else so that other users get the same desktop. Also I can try other things in ~/.xinitrc and then run xinit from the login prompt for testing. Andy


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Cooper Tire Stockholders Approve Merger With Apollo Tyres
Cooper Tire & Rubber Company (NYSE: CTB) today announced that its stockholders voted to approve its pending merger with a wholly-owned subsidiary of Apollo Tyres Ltd (NSE: ApolloTYRE) with more than 74 percent of Cooper's outstanding common shares voted in favor of the transaction. When finalized, the pending merger will result in a strategic business combination that creates the seventh-largest tire company in the world. Approximately 78 percent of Cooper's outstanding common shares were voted, with more than 48 million shares voted in favor of the merger, representing approximately 96 percent of Cooper's voted shares.
"We are pleased stockholders endorsed this compelling transaction, which will create a $6.6 billion leader in the tire industry with a strong global footprint that includes a presence in the world's largest tire market of North America as well as in the fastest growing geographies of India and China," said Cooper Chairman, Chief Executive Officer and President Roy Armes.
Cooper and Apollo announced the proposed merger June 12, 2013 following unanimous approval by the boards of directors of both companies. The pending merger is expected to close by the end of this year.
About Cooper Tire & Rubber Company
Cooper Tire & Rubber Company is the parent company of a global family of companies that specialize in the design, manufacture, marketing, and sales of passenger car and light truck tires. Cooper has joint ventures, affiliates and subsidiaries that also specialize in medium truck, motorcycle and racing tires. Cooper's headquarters is in Findlay, Ohio, with manufacturing, sales, distribution, technical and design facilities within its family of companies located in 11 countries around the world. For more information on Cooper Tire, visit www.coopertire.com, www.facebook.com/coopertire or www.twitter.com/coopertire. FORWARD-LOOKING STATEMENTS This release contains forward-looking statements within the meaning of the federal securities laws. Forward-looking statements are not based on historical facts but instead reflect Cooper's and Apollo's expectations, estimates or projections concerning future results or events. These statements generally can be identified by the use of forward-looking words or phrases such as "believe," "expect," "anticipate," "project," "may," "could," "intend," "intent," "belief," "estimate," "plan," "likely," "will," "should" or similar words or phrases. These statements are not guarantees of performance and are inherently subject to known and unknown risks, uncertainties and assumptions that are difficult to predict and could cause our actual results, performance or achievements to differ materially from those expressed or indicated by those statements. We cannot assure you that any of our expectations, estimates or projections will be achieved.Check Out Our Best Services for Investors
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Saudi Airlines flies to new heights with business intelligence solution from Microsoft
Saudi Airlines, one of the largest airlines in the Middle East, has chosen Microsoft SQL Server 2012, Power BI for Office 365, Windows Server 2012 and Microsoft SharePoint Server 2013 to analyze, visualize and share data in a unified way. By using this Microsoft technology to implement a business intelligence (BI) solution, the airline forecasts better business visibility, more engaged employees and savings of millions of dollars per year. The … Read more »


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Posted: Aug 20, 2014 8:54 AM by Hunter Robinson
Updated: Aug 20, 2014 8:54 AM
Source: WBRZ
BATON ROUGE - Candidate qualifying begins today in Louisiana and U.S. Representative Bill Cassidy (R-Louisiana) was among the first there.
Cassidy will be running against Mary Landrieu for her seat in the U.S. Senate.
We'll have more on the start of candidate qualifying and who's running for what spot, coming up on WBRZ News 2 at Noon.


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Joakim Soria Rumors
Tigers To Exercise Joakim Soria's Option
The Tigers will exercise their $7MM club option on right-hander Joakim Soria, reports Anthony Fenech of the Detroit Free Press (on Twitter).
Soria, 30, was acquired from the Rangers at the trade deadline for the steep price of right-handers Corey Knebel and Jake Thompson, but the results didn't pan out for Detroit. Soria totaled just 11 innings with the Tigers and allowed seven runs (six earned), in part due to an oblique strain that cost him a month of action. He didn't fare any better in the post season, as he was charged with five runs in one inning (two appearances).
Despite the struggles following the trade, Soria was an exceptional bullpen piece with the Rangers this year, pitching to a 2.70 ERA with 11.3 K/9 and 1.1 BB/9 in 33 1/3 innings. His cumulative stat line translated to a 3.80 ERA with a 48-to-6 K/BB ratio in 44 1/3 innings during the regular season.
The Tigers are in desperate need of solidifying their bullpen and will hope that a healthy Soria can assist in accomplishing that goal next season.
AL Central Notes: Aviles, Soria, Burton
As the Royals look to even the score in the World Series against the Giants, other teams in the division are shifting their focus to the offseason. Here are a few notes from the AL Central…
- The Indians are expected to exercise their $3.5MM club option on Mike Aviles rather than pay him a $250K buyout, reports Paul Hoynes of the Cleveland Plain Dealer. The Indians could have reasonably gone either way, as the team has some limited financial flexibility (a topic I highlighted in yesterday's Offseason Outlook). It appears that his versatility will earn him a spot on the 2015 club. The 33-year-old batted .247/.273/.343 in 374 plate appearances with Cleveland last year.
- MLB.com's Jason Beck spoke with Tigers GM Dave Dombrowski, and while the GM didn't specifically say that the team would exercise its $7MM club option on Joakim Soria, he certainly implied as much with his comments: "We still have to make the final decision, but it's a situation where we look at him as being an important acquisition toward this [coming] year also." Dombrowski said that the team views Joe Nathan as its closer next season, though he indicated that Nathan will have to perform up to his previous capabilities in order to hold onto that role.
- The Twins declined their $3.6MM option on Jared Burton earlier today, and Darren Wolfson of 1500 ESPN tweets that the team hasn't expressed an interest in bringing the 33-year-old back, even at a lower rate.
Full Story | Comments | Categories: Cleveland Indians | Detroit Tigers | Jared Burton | Joakim Soria | Mike Aviles | Minnesota Twins
AL Central Notes: Sox, Indians, Tigers' Bullpen
Though the White Sox weren't in a position to add veterans on short-term deals last offseason, GM Rick Hahn feels that with the club closer to contention, that's an avenue worth exploring, writes Dan Hayes of CSNChicago.com. "We're never going to move our eye from those long-term targets being a priority," Hahn said. "At the same time, we may be in a position where some shorter-term deals with some veteran-type players might make some sense in order to get this team closer to where we want to be." The additions of Jose Abreu and Adam Eaton, the progression of Jose Quintana, and the selection of Carlos Rodon in last year's draft have all accelerated Chicago's timeline to contend again. Hayes feels that an outfielder and/or DH would make sense, and he also mentions the bullpen. I think they could look to upgrade behind the dish as well, as Tyler Flowers whiffed at a 36 percent clip last season and is a long shot (to say the least) to repeat his .355 batting average on balls in play. MLBTR's Tim Dierkes recently provided a preview of Chicago's upcoming offseason.
More from the AL Central…
- Once arbitration numbers and league-minimum players are accounted for, the Indians project to have $72-75MM committed to the 2015 roster, MLB.com's Jordan Bastian writes in his most recent inbox piece. The team has typically operated in the $80-85MM payroll range, so there's some degree of financial flexibility. However, Bastian notes that with the large number of contracts spread around the team, Cleveland is limited as to where and how it can upgrade what was a stagnant second-half offense. He feels that third base and right field are the most logical areas to target, noting that the trade market may be "more opportunistic" for the Indians this offseason.
- The Tigers will look to upgrade on a bullpen that melted down and, in the estimation of many, cost them their season in the playoffs, writes James Schmel of MLive.com. However, he cautions not to expect a new closer in 2015. There's a "strong chance" that the team will exercise Joakim Soria's $7MM club option, Schmel writes, and he also notes that Joe Nathan, Al Alburquerque, Bruce Rondon, Blaine Hardy, Kyle Lobstein and Luke Putkonen are expected back. Joba Chamberlain, Phil Coke, Jim Johnson and Joel Hanrahan are likely on the outs. Schmel lists some speculative bullpen targets for Detroit, including Luke Gregerson, Andrew Miller, Neal Cotts and Pat Neshek, and as he notes, the Tigers have been linked to Gregerson in the past and nearly reacquired Miller this past July.
Full Story | Comments | Categories: Chicago White Sox | Cleveland Indians | Detroit Tigers | Joakim Soria
AL Notes: Soria, Indians, Cruz, Bogar
Offense is at a premium this season and Rays manager Joe Maddon doesn't think it will improve any time soon, writes MLB.com's Bill Chastain. "The hitter's at a total disadvantage right now," Maddon said. "And there's no advantages on the horizon. I don't see it. That's why it's going to take a lot of creative thinking. It could be just going back maybe to something that had been done before. I'm not sure. But right now, offense is going south, and it's going to continue going south based on pitching and defense. Everything, data, video, all the information benefits them over offense." Maddon also pointed to improved bullpens throughout baseball as another factor in the depressed offensive numbers.
Here's more from the American League:
- July acquisition Joakim Soria deserves a shot at pitching in high-pressure situations for the Tigers, writes Chris Iott of MLive.com. Soria could be the best relief pitcher the Tigers have and they paid a price to get him, so they should utilize him in the best way possible, Iott argues.
- The Indians led the majors in errors for much of the sesaon, but there likely won't be sweeping changes in Cleveland's infield, writes Paul Hoynes of The Plain Dealer.
- Nelson Cruz reiterated he would like to stay with the Orioles, but extension talks will still wait until after the season, tweets Rich Dubroff of CSNBaltimore.com.
- Tim Bogar is now the clear runaway favorite to be hired as the next Rangers manager, according to Bob Nightengale of USA Today (on Twitter).
Full Story | Comments | Categories: Baltimore Orioles | Cleveland Indians | Detroit Tigers | Joakim Soria | Nelson Cruz | Tampa Bay Rays | Texas Rangers
AL East Notes: Lester, Sox, Lackey, Soria, Yanks, Rays
Red Sox president Larry Lucchino said on WEEI radio's Dennis and Callahan show today that extension talks with ace Jon Lester have been tabled until the offseason. Lucchino reminded that Lester's preference is to avoid negotiating during the season. Lester told reporters, including the Boston Globe's Peter Abraham, that he wasn't surprised to hear Lucchino's comments (Twitter link). However, Rob Bradford of WEEI.com does hear from a source that Lester would be open to signing a midseason deal if the Sox were to offer market value for his services right now. The other possibility for Lester could be a trade, if the Sox fall further back in the East — a concept which Lester admits he has considered (via the Globe's Nick Cafardo). Lester said if it came to that, he wouldn't harbor any ill feelings toward the organization.
Here's more on the BoSox and the AL East…
- Lucchino also said on WEEI that he expects the Red Sox to be active at the trade deadline, though he noted that nearing the deadline with a sub-.500 record is new territory for the Sox. Asked about buying or selling, Lucchino referred to the trade deadline as a "binary process" and implied that the Sox could do some of both.
- Also of note from Lucchino is that the Red Sox plan to engage John Lackey in extension talks after the season. Lackey's Tommy John surgery in 2012 triggered a $500K option for the 2015 season due to an injury clause in his contract. Lucchino is quoted: "I think that there will be some contract negotiations with him probably at the end of the year as well and we'll see what his frame of mind is with respect to longer-term contracts."
- The Blue Jays were in the mix for Joakim Soria before the Rangers traded him to the Tigers, Jon Paul Morosi of FOX Sports reports (Twitter links). Talks never became too serious or progressed to the stage where offers were exchanged, however.
- Morosi also notes that the Yankees are still looking to add a starter to their ranks (Twitter link). Earlier today, he noted that the Yanks scouted Ian Kennedy's last start, though reports from earlier today indicated that the Padres would need to be "overwhelmed" to move him.
- Rays president Matt Silverman tells Marc Topkin of the Tampa Bay Times that there are some "obvious flaws" with the Competitive Balance Lottery, in which the Rays were not awarded a pick yesterday. As Topkin notes, seemingly less-needing teams such as the Cardinals, Orioles and Rockies all received picks. Silverman went on to say, however, that even if the lottery were better constructed, it would "only scratch the surface of the competitive balance issues plaguing baseball."
Full Story | Comments | Categories: Boston Red Sox | Joakim Soria | John Lackey | Jon Lester | New York Yankees | Tampa Bay Rays | Texas Rangers | Toronto Blue Jays
Dave Dombrowski On Joakim Soria, Future Moves
Last night, the Tigers landed one of the top available relievers on the trade market in Rangers right-hander Joakim Soria. While Soria should go a long way towards shoring up Detroit's bullpen, many have wondered if Detroit might continue to work the phones for an additional relief option, particularly given the struggles of left-handers Phil Coke and Ian Krol. I asked Tigers General Manager Dave Dombrowski earlier today if he might go after a southpaw in the next week.
"I don't know I would get into what we're specifically addressing, but we're open-minded to different thought processes. Our bullpen has struggled at times and we want to have people that put up zeroes out there," Dombrowski said. "We've tried a lot of guys at that and we remain open-minded if something makes sense to make us better before the trade deadline."
The Tigers have been in need of bullpen reinforcements for some time and Dombrowski has been focused on Soria "for a while." The GM said that he started chatting with Rangers GM Jon Daniels in June and things picked up more and more with time. And while Soria didn't come cheap — he cost the Tigers promising pitching prospects Jake Thompson and Corey Knebel — he believed that it was a price they could afford to pay thanks to their pitching depth. Soria's affordable $7MM club option also helped Dombrowski to pull the trigger since he will likely be more than just a rental.
"I don't think it made the deal, but it was a real plus for us," the GM said.
Of course, if things went differently for the Tigers this season, they might not have had to make a trade like this at all. Dombrowski acknowledged that he'd be in a "different situation" if Bruce Rondon was with the club. They're also "not counting" on a 2014 return for Joel Hanrahan and the odds were termed as being "highly unlikely."
While Soria has a long history as a successful closer, Dombrowski is insistent that Joe Nathan will continue to pitch in the ninth-inning despite his struggles this season. When asked what it might take for Soria to possible leapfrog Nathan and close out games, the Tigers GM declined to speculate or set expectations for the 39-year-old. For now, Soria is there to help build a better bridge to Nathan and the veteran appears to be all for it. Before pulling the trigger on the deal, Tigers manager Brad Ausmus and pitching coach Jeff Jones asked Nathan and Ian Kinsler about how they feel Soria might fit in with the team. One of the replies they got back was, "Why don't we have him yet?'"
Full Story | Comments | Categories: Detroit Tigers | Joakim Soria | Joel Hanrahan | MLBTR Originals | Newsstand
Reactions To The Joakim Soria Trade
Last night, the Tigers landed right-hander Joakim Soria from the Rangers in exchange for two of their top prospects — right-handers Jake Thompson and Corey Knebel. Detroit has long been said to be in pursuit of relief help, and they've now added one of the top bullpen arms on the market. Here are some reactions to the deal as well as a few additional bits of info about the Tigers' trade talks…
- Ken Rosenthal of FOX Sports tweets that Soria was the Tigers' primary bullpen target, and they didn't pursue former Tiger Joaquin Benoit all that aggressively before landing Soria last night.
- Likewise, ESPN's Jayson Stark tweets that the Tigers were never in on Philadelphia's Jonathan Papelbon all that seriously.
- Soria himself is very excited to join the Tigers, agent Oscar Suarez old FOX Sports' Jon Paul Morosi (Twitter link), specifically mentioning excitement over a chance to win. That excitement isnt surprising for Soria, who didn't have much of an opportunity at the postseason early in his career with the Royals (he wasn't with the club for their recent improvements).
- The Tigers have the prospect depth to add another relief arm if they wish, but it will depend on the asking price, writes Jason Beck of MLB.com. Beck notes that Detroit probably wishes to avoid too much long-term depth to the farm system, but he mentions Chad Qualls as a possibly more affordable option to pursue. Beck also reports that the initial asking price on Soria was higher than the one the Tigers ultimately wound up paying.
- ESPN's Keith Law understands the deal for both Detroit and Texas (Insider subscription required and recommended). While the Tigers paid a steep price, he notes that Soria will be worth about an extra win over the remainder of the season and will be featured in some very high-leverage postseason innings. From Texas' standpoint, they get a raw but projectable 20-year-old in Thompson who needs to learn to get more plane on his fastball and develop a changeup to succeed as a starter, plus a controllable potential seventh- or eighth-inning reliever in Knebel.
- Jim Callis of MLB.com "loves" the Rangers' end of the deal, calling Thompson a potential No. 2 or 3 starter and noting that Knebel has closer upside (Twitter link).
- Soria's value in the postseason could be significant, writes Fangraphs' Dave Cameron. Cameron examines the usage of teams' best relievers in last year's playoffs, noting that while a typical elite reliever throws about five percent of his team's innings during the regular season, that number increases in the playoffs due to more off days and the increased importance of late innings. The Red Sox used Koji Uehara for 9.6 percent of their postseason innings, which translates to about 140 innings during the regular season (a value of 246 innings when accounting for the increased leverage index).
- R.J. Anderson and Jordan Gorosh break down the trade over at Baseball Prospectus (subscription required/recommended). Anderson notes the steep price Detroit paid as well and wonders if the Tigers are done adding relievers. If Dave Dombrowski is serious about truly upgrading the bullpen (which he clearly seems to be), the Tigers should look to add another arm, Anderson opines. Meanwhile, Gorosh feels that Thompson could make an appearance on the back end of B-Pro's offseason Top 101 prospects list, writing that he has the potential to be a "very strong No. 4 starter" and could have been the best pitching prospect in Detroit's system (thereby implying that he likes him better than Robbie Ray).
Full Story | Comments | Categories: Corey Knebel | Detroit Tigers | Joakim Soria | Joaquin Benoit | Jonathan Papelbon | Newsstand | Philadelphia Phillies | San Diego Padres | Texas Rangers
Tigers Acquire Joakim Soria
10:25pm: The Tigers have announced the deal, making it official.
9:23pm: The Tigers have agreed to acquire reliever Joakim Soria from the Rangers in exchange for right-handed pitching prospects Jake Thompson and Corey Knebel, Kyle Bogenschutz of Scout.com was first to report on Twitter. Jon Morosi of FOX Sports confirmed that an agreement is in place (via Twitter).
With the acquisition of Soria, the Tigers now possess both of the Rangers' most recent closers (having signed Joe Nathan as a free agent over the offseason). It appears that Nathan will retain the closer's mantle for the time being, but regardless the team will now have an additional premium arm to throw into high-leverage situations. At the moment, the team owns the fifth-worst relief ERA in baseball.
Soria, a 30-year-old righty, owns a 2.70 ERA with a spectacular strikeout to walk ratio of 11.3 K/9 against only 1.1 BB/9. Indeed, his FIP stands at a miserly 1.07, which handily leads all pitchers who have thrown at least 30 innings. Other ERA estimators (2.25 xFIP; 1.84 SIERA) concur in Soria's excellence to date in 2014.
He is playing out the back end of the two-year, $8MM deal he signed to join the Rangers after Tommy John surgery led to the end of his tenure with the Royals. But Soria also comes with a seemingly reasonable $7MM club option. (That option would increase to $8MM if he finishes 55 games; he is sitting on 32 at present. The contract also includes performance bonuses.)
The return would appear to be substantial. Both Thompson and Knebel rated among Detroit's ten best prospects coming into the year, per Baseball America, with the former landing at fourth and the latter at sixth on BA's list. If anything, their stock has risen since that time.
Thompson, a 20-year-old starter, just earned a promotion to Double-A after posting a 3.14 ERA over 83 innings with 8.6 K/9 and 2.7 BB/9 at the High-A level. Baseball America says he has mid-rotation upside. He has a low-to-mid 90s fastball and promising slider, but profiles as a mid-rotation arm if he can develop a consistent third pitch from amongst his other offerings (curve and circle change).
The 22-year-old Knebel, meanwhile, has already made his big league debut after being selected 39th overall in last year's draft. Though he surrendered six earned runs in 8 2/3 MLB frames, he also notched 11 strikeouts against just three walks and has dominated minor league hitters. He profiles as a potential future closer, says BA, though Detroit had weighed the possibility of trying him as a starter.
With Soria and Huston Street now taken, the relief market now lacks somewhat for obviously available closers. Jonathan Papelbon is surely available, but of course comes with a still-sizable contract. The Padres will presumably listen on Joaquin Benoit, but may want a large return to part with him after dealing Street. And it remains to be seen whether names like Koji Uehara and Steve Cishek could be had.
Full Story | Comments | Categories: Detroit Tigers | Joakim Soria | Newsstand | Texas Rangers | Transactions
Tigers, Pirates, Indians Interested In Benoit
The Tigers, Pirates and Indians are interested in Padres reliever Joaquin Benoit, CBS Sports' Jon Heyman reports. The Padres have already shipped out one reliever with closing experience, trading Huston Street to the Angels, but Heyman writes that Benoit will likely fetch a less attractive return, since teams generally view him as a setup man, not a closer.
Heyman notes that the Tigers or Pirates could use Benoit in the eighth inning, allowing Joe Nathan or Mark Melancon, respectively, to continue to close. (Those two teams are also interested in Joakim Soria of the Rangers, Heyman notes.) It isn't yet clear whether the Indians will be buyers.
Benoit is owed $8MM next year plus $8MM or a $1.5MM buyout in 2016. His 2016 option will vest if he finishes at least 55 games in 2015. Benoit has a 2.04 ERA with 10.4 K/9 and 2.5 BB/9 in 39 2/3 innings with San Diego this year.
Full Story | Comments | Categories: Cleveland Indians | Detroit Tigers | Joakim Soria | Joaquin Benoit | Newsstand | Pittsburgh Pirates | San Diego Padres | Texas Rangers
Tigers, Rangers Discussing Joakim Soria
11:10am: Morosi's colleague, Ken Rosenthal, adds that the Tigers are also interested in Joaquin Benoit but are not actively pursuing Huston Street at this time (Twitter links). The Tigers, according to a rival executive with whom Rosenthal spoke, are interested in adding a reliever that doesn't view himself as a pure closer, suggesting that a new acquisition may not supplant Nathan as the primary ninth-inning option.
9:57am: The Tigers and Rangers are having ongoing discussions about Texas closer Joakim Soria, reports Jon Paul Morosi of FOX Sports 1 (on Twitter).
The back end of the bullpen was a worry that Detroit GM Dave Dombrowski hoped not to have when he signed Joe Nathan this offseason, but the former Twins/Rangers stopper hasn't pitched like himself in 2014. Nathan has posted a 5.61 ERA in 33 2/3 innings with the Tigers, and while his strikeout rate remains strong (9.4 K/9), his walks are up and his fastball is averaging a career-worst 91.7 mph. That velocity dip could have something to do with the increase in his home run rate and decrease in swinging-strike rate as well.
Soria, on the other hand, has been excellent in his first full season back from Tommy John surgery. The former Royals closer has turned in the best strikeout and walk rates of his career (11.9 K/9, 1.2 BB/9) en route to a 2.67 ERA and 16 saves in 30 1/3 innings of relief work.
An acquisition of Soria would give the Tigers an improved ninth-inning option not only for 2014 but also 2015, as his contract contains a $7MM club option ($500K buyout). Beyond that, Soria is highly affordable in 2014, as he's earning just a $5.5MM salary, of which $2.19MM remains.
This would be the second straight season in which the Tigers have acquired bullpen help, should a deal come to fruition. However, the asking price on Soria will undoubtedly be higher than last year's asking price for Jose Veras. Detroit sent Danry Vasquez and David Paulino to Houston in order to complete that deal.
The Rangers have reportedly been receiving a good deal of interest in their bullpen arms, and they've already shown a willingness to move veteran pieces by sending Jason Frasor to the Royals this week.
Full Story | Comments | Categories: Detroit Tigers | Huston Street | Joakim Soria | Joaquin Benoit | Newsstand | San Diego Padres | Texas Rangers


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First Age
Where Love Is: 10. Nine
winter, 470
I hate your Oath. I do not pretend to feign surprise that you were capable of swearing it. I know you too well for that. But its legacy is cruel: forever binding us to the past, with all its wounds and anguishes, perpetuating with words what we might have conquered with time. And for what? The Silmarils, created things, fair indeed, but temporal. Jewels and a dead man's grief.
I want to love you, but another man comes between us: a Son of Fëanor, proud and rash, bent on vengeance. A man who has sworn his life away with words binding as the vows of our love, invoking curse, not blessing. When I reach out for you, it is this man whom I find, this living anachronism who masquerades as my beloved, and moves him to ill deeds.
Swear a new oath, one I can swear beside you. Other memory consumes your thoughts, I know: the cliffs of Thangorodrim like a purgatory of the mind, a place that neither of us shall ever truly leave. You, who suffered so excruciatingly, and I who beheld your dangling frame in anguish, who cut through sinew and bone to free you, and felt the pain I inflicted in the core of my own heart. If we are to war, let this be our rallying oath--to avenge you and all of us, and to ruin the prisons of the Enemy forever.
Beloved... I wonder what will come of all this--Doriath and the sons of Fëanor, you and I--and cannot find an answer.


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PC Connection, Inc. Reports Second Quarter Results
Overall annualized sales productivity was largely unchanged in the second quarter of 2011 compared to the second quarter of 2010. Sales productivity in the SMB segment increased by 5%, but was offset by decreases in the Large Account and Public Sector segments of 5% and 1%, respectively. On a consolidated basis, the total number of sales representatives was 630 at June 30, 2011, compared to 588 at June 30, 2010, and 634 at March 31, 2011.
Total selling, general and administrative expenses for the quarter increased year over year by 15%, or $7 million, and increased as a percentage of net sales from 9.9% for the second quarter of 2010 to 10.6% for the second quarter of 2011. The year-over-year increase was attributable to investments in solutions sales capabilities, increased variable compensation associated with improved gross profit, increased marketing expenditures, and the inclusion of ValCom's operating costs for the quarter.
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About PC Connection, Inc.
PC Connection, Inc., a Fortune 1000 company, operates through five sales subsidiaries: PC Connection Sales Corporation, MoreDirect, Inc., GovConnection, Inc., PC Connection Express, Inc., and Professional Computer Center, Inc. d/b/a ValCom Technology, headquartered in Merrimack, NH, Boca Raton, FL, Rockville, MD, Portsmouth, NH, and Itasca, IL, respectively. All five companies can deliver custom-configured computer systems overnight from PC Connection Services' ISO 9001:2008 certified technical configuration lab at its distribution center in Wilmington, OH. Investors and media can find more information about PC Connection, Inc. at http://ir.pcconnection.com. PC Connection Sales Corporation (800-800-5555), the original business of PC Connection, Inc. serving the small- and medium-sized business sector, is a rapid-response provider of IT products and services. It offers more than 180,000 brand-name products through its staff of technically trained sales account managers and catalog telesales representatives, catalogs, and publications, and its website at www.pcconnection.com. MoreDirect, Inc. (561-237-3300), www.moredirect.com, provides corporate technology buyers with a comprehensive web-based e-procurement solution and in-depth IT supply-chain expertise, serving as a one-stop source by aggregating more than 300,000 products from the inventories of leading IT wholesale distributors and manufacturers. MoreDirect's TRAXX™ system is a seamless end-to-end interface that empowers clients to electronically source, evaluate, compare prices, and track related technology product purchases in real-time. GovConnection, Inc. (800-800-0019) is a rapid-response provider of IT products and services to federal, state, and local government agencies and educational institutions through specialized account managers, catalogs, and publications, and online at www.govconnection.com. PC Connection Express, Inc. (888-800-0323) is a rapid-response provider of computer products and consumer electronics to home, home office, and small office users. Customers can purchase the best-known brands in the industry online at www.pcconnectionexpress.com or order by calling a trained sales specialist. The subsidiary includes the MacConnection brand (800-800-2222), one of Apple's largest authorized online resellers at www.macconnection.com. Professional Computer Center, Inc. d/b/a ValCom Technology (630-285-0500), www.valcomtechnology.com, provides technology services to medium-to-large corporate organizations utilizing its proprietary cloud-based IT service management software, WebSPOC™. Through its experienced technical service personnel ValCom Technology provides network, server, storage, mission-critical onsite support, installation, and host of lifecycle services. pccc-gCheck Out Our Best Services for Investors
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This is the time of year that we are indulging more than ever. Your favorite aunt arrives with the best cookies you've ever had. Your mom makes your favorite cake and brings it over for all to enjoy. Co-workers bring all of their best holiday creations to work daily. It is wonderful until we start to make our holiday resolutions. Our number one resolution will be to lose weight.
There is nothing wrong with eating your favorite things in moderation. In the mean time you should eat healthy. The odd thing about buying healthy foods is that they are more expensive. You would think if it has less sugar, fat and preservatives, it would be cheaper. Not true! You can still eat healthy and stay in your budget. Just follow a few tricks. Watch sales closely. You can keep frozen fruits and vegetables in your freezer all the time. Buy bag salads when they are on sale. Buy fruit in bags or bulk. Stay away from cans as much as possible because they have additives and not as much nutrients as fresh or frozen.
Another great idea is to visit you local farmer's market and stock up. This is the time of year for collards, turnips and winter squash. Apples are also in abundance at the market now. You will be surprised at all the things available. A great place to check out in our area is Pee Dee Farmers market in Florence or McCleod Farms in McBee. You can even order online baskets. They have the best peaches!
After a day of eating cookies, cake, chocolate and whatever other delicious treats are offered that day, coming home to a healthy meal may be just the ticket to keep you feeling good and not run down. Make a big salad of whatever raw vegetables you have on hand. Some great ideas are lettuce or baby spinach leaves, tomatoes, onions, peppers, cucumbers and carrots. Top with grilled chicken strips and a light dressing such as an oil and vinegar based dressing.
Another idea would be to use your crock pot. Before you leave for work, put a whole chicken in a lightly oiled crock pot. Sprinkle salt an pepper over the meat and add about a cup of either water or low sodium chicken broth to the pot. Cook on low all day and you will have a perfectly cooked chicken to bone and use in a variety of healthy dishes. Chicken salad can be made with fat free mayonnaise, and pickle relish (celery if desired). Eat on whole wheat bread and you have a very healthy meal. You can also shred the chicken and add a little salsa to it. Wrap in a tortilla with shredded lettuce ant tomatoes. This is a very tasty and nutritious meal.
Enjoy the holiday season and all of the extra goodies but most importantly don't overdue. Take care of yourself and your family. Being healthy and fit is the best gift we can give to ourselves and our family. Accomplishing this without killing our budget is icing on the cake!


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Kim Jong-Un Tired Of Being World Punchline
Description:Lil Kim (aka Kim Jong-Un) is less than thrilled with South Korea's celebrated rocket launch. The North Korean leader believes the UN sanctions unfairly target his country. (1:12)
Source: NewsFix DFW
Tags: cw33 , kim jong-un , lil kim , nightcap