Sec22p Export from the Endoplasmic Reticulum Is Independent of SNARE Pairing

Abstract

Molecularly distinct sets of SNARE proteins localize to specific intracellular compartments and catalyze membrane fusion events. Although their central role in membrane fusion is appreciated, little is known about the mechanisms by which individual SNARE proteins are targeted to specific organelles. Here we investigated functional domains in Sec22p that direct this SNARE protein to the endoplasmic reticulum (ER), to Golgi membranes, and into SNARE complexes with Bet1p, Bos1p, and Sed5p. A series of Sec22p deletion mutants were monitored in COPII budding assays, subcellular fractionation gradients, and SNARE complex immunoprecipitations. We found that the N-terminal "profilinlike" domain of Sec22p was required but not sufficient for COPII-dependent export of Sec22p from the ER. Interestingly, versions of Sec22p that lacked the N-terminal domain were assembled into ER/Golgi SNARE complexes. Analyses of Sec22p SNARE domain mutants revealed a second signal within the SNARE motif (between layers ؊4 and ؊1) that was required for efficient ER export. Other SNARE domain mutants that contained this signal were efficiently packaged into COPII vesicles but failed to assemble into SNARE complexes. Together these results indicated that SNARE complex formation is neither required nor sufficient for Sec22p packaging into COPII transport vesicles and subsequent targeting to the Golgi complex. We propose that the COPII budding machinery has a preference for unassembled ER/Golgi SNARE proteins.In eukaryotic cells, most intracellular membrane fusion processes are mediated by a related family of small proteins, called SNARE 1 (soluble NSF attachment protein receptors) proteins (1, 2). SNAREs share a conserved heptad repeat coiled coil sequence, called the SNARE domain, that is typically adjacent to a C-terminal-membrane bound region (3). Specific sets of SNARE domains assemble into parallel four-helix bundles forming the stable core of a SNARE protein complex (4 -6). Current membrane fusion models theorize that cognate sets of SNARE proteins provided from opposing membranes assemble into "trans" SNARE complexes and drive membrane bilayers together during fusion (2, 7).The assembly of cognate SNAREs has also been proposed to define the compartmental specificity of membrane fusion based on experiments showing that only specific combinations of SNARE proteins catalyze in vitro fusion when reconstituted into synthetic proteoliposomes (8 -10). However, this proposal remains debated given the promiscuity of SNARE interactions in vitro (11-13), functional redundancy in vivo (14, 15), and requirements for individual SNARE proteins in multiple intracellular membrane fusion steps (16). These and other observations suggest that additional spatial and temporal determinants in SNARE proteins contribute to the specificity of the membrane fusion (2). The N-terminal domains of SNARE proteins display significant diversity and appear to govern SNARE protein activity and distribution. For example, the N-terminal domain of the syntaxin-like SNARE prot...

Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

“…To visualize vacuoles, cells Thus, unlike the Nyv1p-LD, when the longin domains of Sec22p and Ykt6p are tethered to membranes via the Snc1p transmembrane domain, they do not seem to convey any dominant protein sorting signals. However, we cannot exclude the possibility that the longin domain of Sec22p is required for COPII-mediated export from the ER (Miller et al, 2003;Mossessova et al, 2003;Liu et al, 2004). regions of the longin fold, and both are buried in the core of the protein, whereas the third sequence, Y 31 GTI 34 , is located in a surface-exposed and highly dynamic region between ␤B and ␣A ( Figure 6, A and B).…”

Genetic Analysis of Yeast Sec24p Mutants Suggests Cargo Binding Is Not Co-operative during ER Export

“…However, our observed rescue of both the B-and C-site Sec24p mutants with a construct that would not be competent for ER export, sec22-ΔN3, suggests that the most important function of Sec22p in these cells is as a retrograde SNARE receiving transport vesicles from the Golgi. Consistent with this model, N-terminal truncations in Sec22p that are not packaged into COPII vesicles are also capable of complementing a sec22Δ mutant (22). Although non-specific or bulk flow transport of truncated Sec22p may permit a limited amount of ER export that might complement a lack of Sec22p, such a low level of Sec22p flux is difficult to reconcile with a rescue of a Bet1p (or other essential cargo) defect.…”

“…Since this truncation removes the conformational epitope recognized by Sec24p it should no longer be recruited into COPII vesicles and remain ER retained. Indeed, similar N-terminal truncations of Sec22p largely retained their ability to interact with the cognate ER-to-Golgi SNAREs but were excluded from COPII vesicles (22). Overexpression of sec22-ΔN3 was able to rescue the sec24-L616W B-site mutant, suggesting that the Sec22p SNARE domain alone is sufficient for rescue and that uptake into COPII vesicles is not required for this in vivo complementation.…”

“…Biochemical and structural experiments have indicated that the N-terminal longin domain plus a short segment of the SNARE domain are required for Sec22p to bind the interface between Sec23p and Sec24p (14,22). A truncated form of Sec22p lacking most of the Cterminal half of its SNARE domain, sec22-ΔS2, is deficient in binding its cognate ER-toGolgi SNARE proteins Bet1p, Bos1p, and Sed5p but is still captured into COPII vesicles in vitro since it retains the conformational ER export epitope (22). If Sec22p rescues the B-site simply by binding the C-site and thereby stabilizing the B-site, sec22-ΔS2 should suffice to rescue the Sec24p B-site mutants.…”

“…SMY2_425 was constructed by cloning a 2.6kb fragment containing the SMY2 gene and a 5' UTR extending up to the neighboring ORF into the SpeI/XhoI sites of pRS425. The Sec22 SNARE domain partial deletion was constructed by restriction digesting Sec22-SE-B (22) with MfeI and religating the plasmid, resulting in a 1.5kb fragment consisting of the SEC22 gene, including 5' and 3' UTRs, with a deleted segment from nucleotides 489 to 555cloned into the BamHI/ EcoRV sites of pRS313, creating pRB058. The construct was subcloned into the SpeI/XhoI sites of pRS425 to produce pRB059.…”