Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting
© Byers et al; licensee BioMed Central Ltd. 2009
Received: 18 September 2008
Accepted: 19 June 2009
Published: 19 June 2009
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 . 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  and the chloroplast outer envelope in plants . 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 . 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 . 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., , Brambillasca et al.,  and Wattenberg et al.,  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 . 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 .
We have previously shown that when TA1 or TA2 are encoded as part of SLMAP1 they direct it to subcellular membrane structures  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  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 . 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
Tail anchors in SLMAP target distinct subcellular membranes
Disruption of the cytoskeleton and SLMAP localization
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 . 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
Hydrophobic profiles of the TAs in SLMAP
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) . 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 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 . 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 . 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 . 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  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 .
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 .
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.,  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 . 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 . 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 with the Eisenberg algorithm using a window size of 9 and the relative weight for the window edges was 100%. .
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/AT A) was created using 5' (GGA AAT AAT AT A CCC TGG CCC) and 3' (GGG CCA GGG TA T ATT ATT TCC). The R27/I mutant (AGA/AT A) was created using 5' (CCA GGT CTG GCC AT A GCT TCT CCG TG) and 3' (CA CGG AGA AGC TA T 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 AT A GCT TCT CCG TG) and 3' (CA CGG AGA AGC TA T GGC CAG ACC TGG)). The P4/L mutant (CCC/CTC) was created using 5' (ATA CCC TGG CT C 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 CT T CT C 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 CT A 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 CT A 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 , 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 . 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., . 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 . 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.
This work was supported by an operating grant from the Heart and Stroke Foundation of Ontario (#T5821) to BST.
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