Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.Vesicles transport biosynthetic cargo and lipids between different compartments of the endomembrane system. Formation of the transport vesicles requires adaptors, coat proteins, and regulatory GTPases of the Arf1/Sar1 family. The initial contact between a vesicle and its target membrane requires Rab GTPases and tethers, which are in most cases multisubunit complexes. Rab GTPases, which cycle between an inactive GDP-and active GTP-bound state, and tethers coordinate the assembly of SNARE proteins on vesicle and target membrane into a four-helix bundle, which ultimately drives bilayer fusion (1).The Dsl1 tethering complex functions in fusion of Golgiderived vesicles at the ER 4 membrane and consists of the three subunits Dsl1, Dsl3/Sec39, and Tip20. It forms a stable complex with the ER SNAREs Sec20, Ufe1, and Use1 (2, 3). In addition, Dsl1 and Tip20 are linked to the coatomer, which implies a role in the recognition and/or uncoating of the COPI vesicle (4 -7). In agreement with this, Dsl1 depletion leads to a massive accumulation of COPI-coated vesicles (8).The Dsl1 complex is closely linked to the SNAREs Ufe1, Use1/Slt1, Sec20, and Sec22, which are required for fusion at the ER membrane (9 -12). The R-SNARE Sec22 is generally accepted as the v-SNARE on COPI vesicles. However, Sec22 has not been previously identified as part of the Dsl1 complex and is dispensable for yeast survival. In addition, it can be functionally replaced by the R-SNARE Ykt6 in anterograde (13) and potentially also in retrograde transport. Ykt6, which lacks a transmembrane domain and thus is unlikely to function as the sole v-SNARE, is found in multiple SNARE complexes at the Golgi, endosomes, and the vacuole (10, 14).Here, we present additional insights into the interactions and functions of the Dsl1 complex. We show that the two R-SNAREs Sec22 and Ykt6 are associated with the Dsl1 complex, with Sec22 being the preferred subunit. Only Ykt6 is sensitive to Sec18/NSF, whereas the remaining interaction between SNAREs and the Dsl1 complex is unaffe...

The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1⌬ and yet3⌬ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1⌬ or yet3⌬ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.

Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell-mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high affinity receptor, FcεRI, on the cell surface. Here we use the endosomal v-SNARE, VAMP8, and the lysosomal hydrolase β-hexosaminidase (β-Hex), each C-terminally fused to super-ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin-tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with TIRF microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β-Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F-actin with jasplakinolide inhibits antigen-stimulated exocytosis but is not additive with S25N Rab11-mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-tophenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.