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Export of transmembrane proteins from the endoplasmic reticulum (ER) is driven by directed incorporation into coat protein complex II (COPII)-coated vesicles. The sorting of some cargo proteins into COPII vesicles was shown to be mediated by specific interactions between transmembrane and COPII-coat-forming proteins. But even though some signals for ER exit have been identified on the cytosolic domains of membrane proteins, the general signaling and sorting mechanisms of ER export are still poorly understood. To investigate the role of cargo protein oligomer formation in the export process, we have created a transmembrane fusion protein that – owing to its FK506-binding protein domains – can be oligomerized in isolated membranes by addition of a small-molecule dimerizer. Packaging of the fusion protein into COPII vesicles is strongly enhanced in the presence of the dimerizer, demonstrating that the oligomeric state is an ER export signal for this membrane protein. Surprisingly, the cytosolic tail is not required for this oligomerization-dependent effect on protein sorting. Thus, an alternative mechanism, such as membrane bending, must account for ER export of the fusion protein.
In eukaryotic cells, transmembrane proteins destined for the secretory pathway or the plasma membrane are first inserted into the endoplasmic reticulum (ER). From there, they travel to the Golgi apparatus in membrane vesicles covered with the coat protein complex II (COPII) coat [1-3], which minimally comprises five proteins: the small GTPase Sar1 and the two protein complexes Sec23/24 and Sec13/31. Upon activation by guanosine triphosphate (GTP) binding, Sar1 recruits Sec23/24 to the ER membrane; it then interacts with Sec13/31, directing it to the membrane. Sec13/31 polymerizes the COPII coat and composes the outermost layer of the emerging vesicle [4-7].
Protein transport out of the ER was initially proposed to be non-selective, with ER-resident proteins maintaining their steady-state distribution via selective retrieval [6, 8-10]. However, quantitative comparison of the contents of COPII-coated vesicles to those of the ER membranes revealed that many cargo proteins were highly enriched in the vesicles [11, 12]. Amino acid motifs required for concentrative packaging have been identified in the sequence of several cargo proteins, as have the specific interactions between these motifs and components of the COPII coat [13, 14]. Additional studies have identified transmembrane receptor proteins that link soluble cargo proteins in the lumen of the ER to the cytosolic coat proteins . Large cargo proteins (e.g. collagen), which do not fit in a standard COPII vesicle, need additional coat-binding or -regulating proteins (e.g. TANGO1, cTAGE5 and Sedlin) to be incorporated [6, 16-19]. Thus, uptake into COPII vesicles employs a specific sorting and concentration process in vitro and in vivo [20-22], and direct protein–protein binding interactions provide one compelling mechanism for mediating protein sorting in the ER.
To date, the majority of work has focused on the signal-mediated interactions of cargo proteins with the COPII coat that direct cargo into COPII vesicles [13, 23-28]. Cargo proteins can directly interact with one of three binding sites on the COPII coat component, Sec24, with an additional site in the human C and D isoforms [29-33]. Several Sec24-binding motifs have been discovered on a variety of eukaryotic membrane proteins including dihydrophobic [13, 34], diacidic [26, 35], triple arginine  and aromatic motifs [25, 26].
The majority of ER-exported membrane proteins, however, carry no known export signal in their sequence [6, 27]. Thus, either new signals remain to be identified or something else drives their recruitment into COPII vesicles. As many membrane proteins form oligomers prior to export from the ER , combinatorial signals (i.e. oligomeric signals composed from many weakly interacting sequences) have been postulated to link oligomerization to efficient export . Indeed, for a yeast COPII-binding cargo receptor protein and its mammalian homolog, oligomerization is required for its export from the ER [38, 39].
In this article, we report the construction of a model transmembrane fusion protein that can be oligomerized in isolated microsomal membranes by a chemical dimerizer. We find that uptake of the oligomerized protein into COPII vesicles in vitro is strongly enhanced and independent of the cytosolic domain. Apparently, the oligomeric status of a membrane protein alone can promote its exit from the ER independent of any known export signal. We propose that protein oligomerization can generate a local membrane curvature that promotes vesicle formation, and thus the oligomeric form is preferentially packaged into transport vesicles without direct protein–protein binding interactions.
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We have constructed a chimeric, oligomerization-competent membrane protein, FKBP-TMD-FF (FT-FF), to investigate the influence of cargo protein oligomerization on ER export. The cytosolic tail of the construct contains two distinct sequence motifs that have been implicated in promoting transport of membrane proteins out of the ER, neither of which significantly contributed to the packaging of the fusion protein into COPII vesicles. The C-terminal diphenylalanine (FF) motif is also present in the cytosolic tail of some p24 proteins [13, 59] and of ERGIC-53, where mutation leads to a decreased ER export of the protein in vivo, concurrent with diminished in vitro binding to COPII proteins [14, 25].
We have established by three independent techniques that binding of Sec24 to the Prm8-derived cytosolic tail sequence of FT-FF indeed depends on the C-terminal phenylalanines. Still, FT-FF and FT-AA are packaged into COPII vesicles with the same efficiency in vitro, and addition of dimerizer dramatically enhances packaging of both fusion proteins (Figures 4 and 6). Another potential export motif identified in some proteins, F/Yx4F/Y [13, 45], is present in the Prm8 sequence: CGGYWRQEYPGVDEFF. We have not conducted binding experiments in which this sequence was modified, but because FT-R and FT-S both showed dimerizer-dependent budding to the same extent as FT-FF (Figure 6B), we assume that this sequence does not mediate the degree of packaging into COPII vesicles in our system.
The hypothesis that oligomerization signifies the culmination of protein synthesis and folding in the ER, and simultaneously a signal to initiate export, was formulated some time ago [22, 37, 60, 61]. Numerous examples of proteins that might act in this way have been documented, such as the T-cell receptor , influenza virus hemagglutinin (a homotrimer) , ERGIC-53 (a homohexamer) [64, 65], vesicular stomatitis virus G protein (a homotrimer) [64, 66], sodium-dependent neurotransmitter transporters , the yeast Vig4/Gog5p transporter (a homodimer) [68, 69] as well as yeast Erv41p/Erv46p  and Emp46p/Emp47p heterodimers . Similarly, the N-acetylglucosamine-1-phosphotransferase (PT) α/β-subunit precursor protein can be transported only if a combinatorial sorting motif forms an epitope in the heterodimer, which can be recognized by the COPII machinery, suggesting that oligomerization can generate export motifs missing in the single subunits  thereby indicating the completion of protein folding. Our findings now provide a confirmation of this hypothesis for a model protein and constitute a novel example for a protein without a known (or indeed possible) cytosolic export signal.
We first assumed that formation of oligomers would lead to the presentation of the C-terminal cytosolic tails to the COPII coat as a linked oligomeric array and thus increase the avidity of the interaction between coat and cargo proteins as shown for the γ-aminobutyric acid (GABA) transporter 1 (GAT 1) [72, 73]. However, our results provide evidence for a novel effect of oligomerization as the cytosolic tail of the fusion protein seems to have no influence on the efficiency of export from the ER membranes: both COPII-binding and non- (i.e. weak-) COPII-binding constructs (FT-AA, Figure 5; FT-R, -S; data not shown) show dimerizer-dependent ER exit (Figure 6A, B). Our data thus support the hypothesis that the oligomeric state of a membrane protein itself can act as an ER export signal without the need to bind to the cytosolic COPII components. The possibility remains that oligomers interact with a cargo receptor via the transmembrane or lumenal domains that would serve as a link to the COPII coat, but we have no reason to believe that either the HA epitope tags or the FKBP domains would have such a cargo receptor in yeast.
Another mechanism that deserves consideration involves dominant retention. In some cases, formation of hetero-oligomers masks retrieval and/or retention signals on subunits in a complex and thus moves the steady-state location of a protein to the Golgi apparatus and beyond: the chloride channel Kir6.2  and connexin-43 contain cytosolic arginine motifs that are masked , the IgE receptor  and the lipid phosphatase Sac1 contain dilysine motifs that are masked , the T-cell receptor has charged residues in its transmembrane domains that must be properly paired [61, 78] and the asialoglycoprotein receptor subunits are retained by ER lumenal pentapeptide sequences unless they are joined together in the dimer . We believe that this does not occur with the FKBP constructs because the α-FT-FF fusion protein is transported rapidly and efficiently out of the ER in vivo, suggesting that there is no dominant retention signal on the FKBP or TMD sequences (Figure 2B). Moreover, we do not observe major cross-links of FT-FF to any other protein in the absence of dimerizer (Figure 5). We cannot, however, exclude that interaction of the prosequence with the cargo receptor Erv29 could outbalance retentive chaperone binding in vivo.
Another explanation for our observations is that oligomerization of our model protein changes its physical properties such that it becomes enriched in COPII budding sites, for example, in regions of curved membrane or in regions of different lipid composition [80-82]. This mechanism is known to act in other steps in vesicular trafficking where oligomerization-induced transport has been demonstrated. For instance, the binding of antigen by the B-cell receptor at the cell surface leads to its oligomerization in lipid rafts (probably brought about by the altered physical properties of the oligomer) and internalization , and a similar mechanism is postulated for the Fcε receptor . In early endosomes, targeting of Fc and transferrin receptors toward lysosomes is enhanced by oligomerization ; a lipid-based sorting mechanism may be at work here, too . Furthermore, it is well known that proteins and protein complexes can force lipid membranes to bend, e.g. rigid proteins exhibiting domains of intrinsic curvature like the banana-shaped Bin-Amphiphysin-Rvs (BAR) domain, or the COPII coat protein complexes Sec23/24 and Sec13/31, which have a concave membrane contact surface after polymerization [87-89]. Copic et al. have recently shown that Sec13 is dispensable for the generation of COPII vesicles when non-essential membrane cargo proteins are depleted, proposing that asymmetrical membrane cargo (e.g. glycosyl-phosphatidylinositol-anchored or p24-family proteins with their mass at the luminal side of the ER membrane) might cause an inward, concave curvature of the membrane (into the ER lumen) . This inward curvature resisted the formation of convex COPII buds and could only be overcome by a fully functional COPII coat, rigid enough to enforce a concave, outward curvature and thus the generation of a transport vesicle [90, 91]. The antagonism between membrane curving protein coats that drive vesicle formation and energy barriers (generated for instance by lumenally oriented membrane proteins) that oppose it were recently reviewed by Stachowiak et al. .
Intriguingly, for our FT fusion proteins we observed that packaging of the lower band species (which we believe are inserted into the membrane in a type II orientation) into COPII vesicles often became less efficient as the concentration of dimerizer was increased (this is especially well visible for FT-FF in Figure 4 and for FT-S in Figure 6B). This suggests that a membrane curvature model provides the simplest explanation for the oligomerization-induced enhancement of COPII packaging that we observe. We hypothesize that the lower mobility FT species, correctly inserted in the type I orientation, forms an oligomer that fits better into or even supports formation of the convex COPII buds (Figure 7). In contrast, the higher mobility type II-oriented oligomer may locally impart a concave shape on the ER membrane that is not conducive to COPII vesicle formation and this form is therefore excluded from COPII vesicles.
Figure 7. One hypothetic model: FT-FF influences membrane bending upon dimerization. A) FT-FF is randomly inserted into the ER membrane in type I and type II orientation (lower and faster mobility species in the gels, respectively, see, e.g. Figure 5). B and C) Addition of the dimerizer AP20187 results in polymerization of the respective FT-FF species. We hypothesize a complex conformation that strongly imparts membrane bending (depicted as triangular FKBP domains): The type II-oriented oligomer favors a concave membrane curvature opposing vesicle generation (B), whereas the type I-oriented oligomer induces convex membrane bending thereby facilitating COPII vesicle formation (C). HA2 tag and glycans of the protein construct are omitted for clarity.
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Whichever mechanism causes the increased packaging of oligomeric proteins, it is tempting to envisage situations in which the degree of oligomerization of a protein is used to control its export from the ER in vivo. First, correct folding and maturation of a protein are required for oligomerization (see above), ensuring that only correctly folded proteins enter the secretory pathway. Second, membrane protein oligomerization in the ER can be induced by the binding of a ligand to the lumenal domain, in analogy to the ligand-induced dimerization of cell surface receptors. In this manner, lumenal cargo proteins could bind to their transmembrane receptors via protein or glycan residues, bring about their oligomerization and COPII binding and thus initiate their own uptake into ER to Golgi transport vesicles. Such a mechanism has been described in the Golgi apparatus for the induction of COPI vesicle formation by the KDEL receptor, Erd2 . Third, a conformational change in a protein could lead to an increase in affinity for a specific domain of the ER membrane (e.g. a lipid raft), which may then lead to its oligomerization owing to its increased concentration in such a domain.