Sec22p export from the endoplasmic reticulum is independent of SNARE pairing

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 "profilin-like" 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.     

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.     

Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability

Tethering factors mediate initial interaction of transport vesicles with target membranes. Soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) enable consequent docking and membrane fusion. We demonstrate that the vesicle tether conserved oligomeric Golgi (COG) complex colocalizes and coimmunoprecipitates with intra-Golgi SNARE molecules. In yeast cells, the COG complex preferentially interacts with the SNARE complexes containing yeast Golgi target (t)-SNARE Sed5p. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a, the mammalian homologue of Sed5p. Moreover, fluorescence resonance energy transfer reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady-state levels of the intra-Golgi SNARE complex. Finally, overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t-SNARE Syntaxin5a/Sed5p and enhancing the stability of intra-Golgi SNARE complexes.     

The Aspergillus nidulans syntaxin PepAPep12 is regulated by two Sec1/Munc‐18 proteins to mediate fusion events at early endosomes,late endosomes and vacuoles

Syntaxins are target‐SNAREs that crucially contribute to determine membrane compartment identity. Three syntaxins, Tlg2p, Pep12p and Vam3p, organize the yeast endovacuolar system. Remarkably, filamentous fungi lack the equivalent of the yeast vacuolar syntaxin Vam3p, making unclear how these organisms regulate vacuole fusion. We show that the nearly essential Aspergillus nidulans syntaxin PepA^Pep12, present in all endocytic compartments between early endosomes and vacuoles, shares features of Vam3p and Pep12p, and is capable of forming compositional equivalents of all known yeast endovacuolar SNARE bundles including that formed by yeast Vam3p for vacuolar fusion. Our data further indicate that regulation by two Sec1/Munc‐18 proteins, Vps45 in early endosomes and Vps33 in early and late endosomes/vacuoles contributes to the wide domain of PepA^Pep12 action. The syntaxin TlgB^Tlg2 localizing to the TGN appears to mediate retrograde traffic connecting post‐Golgi (sorting) endosomes with the TGN. TlgB^Tlg2 is dispensable for growth but becomes essential if the early Golgi syntaxin SedV^Sed5 is compromised, showing that the Golgi can function with a single syntaxin, SedV^Sed5. Remarkably, its pattern of associations with endosomal SNAREs is consistent with SedV^Sed5 playing roles in retrograde pathway(s) connecting endocytic compartments downstream of the post‐Golgi endosome with the Golgi, besides more conventional intra‐Golgi roles.     

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.     

Multiple SNARE interactions of an SM protein: Sed5p/Sly1p binding is dispensable for transport

Sec1/Munc18 (SM) proteins are central to intracellular transport and neurotransmitter release but their exact role is still elusive. Several SM proteins, like the neuronal N-Sec1 and the yeast Sly1 protein, bind their cognate t-SNAREs with high affinity. This has been thought to be critical for their function. Here, we show that various mutant forms of Sly1p and the Golgi-localized syntaxin Sed5p, which abolish their high-affinity interaction, are fully functional in vivo, indicating that the tight interaction of the two molecules per se is not relevant for proper function. Mutant Sly1p unable to bind Sed5p is excluded from core SNARE complexes, also demonstrating that Sly1p function is not directly coupled to assembled SNARE complexes thought to execute membrane fusion. We also find that wild-type Sly1p and mutant Sly1p unable to bind Sed5p directly interact with selected ER-to-Golgi and intra-Golgi nonsyntaxin SNAREs. The newly identified, direct interactions of the SM protein with nonsytaxin SNAREs might provide a molecular mechanism by which SNAREs can be activated to engage in pairing and assemble into fusogenic SNARE complexes.     

COG6 Interacts with a Subset of the Golgi SNAREs and Is Important for the Golgi Complex Integrity

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The conserved oligomeric Golgi (COG) complex has been specifically implicated in the tethering of retrograde intra‐Golgi vesicles. Using yeast two‐hybrid and co‐immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27 and SNAP29. Interaction with SNAREs is accomplished via the universal SNARE‐binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE‐binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6–SNARE interactions are important for both COG6 localization and Golgi integrity .     

Analysis of Sec22p in endoplasmic reticulum/Golgi transport reveals cellular redundancy in SNARE protein function

Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. In sec22Delta strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of the sec22Delta mutation with a conditional ykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization.     

ER-Golgi transport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit, Sec24p

Selective cargo capture into ER-derived vesicles is driven by the Sec24p subunit of the COPII coat, which contains at least three independent cargo-binding sites. One of these, the "A-site," interacts with a NPF motif found on the SNARE, Sed5p. We have characterized the Sec24p-Sed5p interaction through mutation of the putative ER export motifs of Sed5p and the cargo-binding A-site of Sec24p. Mutational analysis of Sed5p suggests that the NPF motif is the dominant ER export signal. Mutation of the NPF binding pocket on Sec24p led to a dramatic reduction in the capture of Sed5p into COPII vesicles, whereas packaging of other ER-Golgi SNAREs was normal. Of all the cargoes tested, only Sed5p was depleted in vesicles made with Sec24p A-site mutants. Surprisingly, vesicles generated with the mutant Sec24p were unable to fuse with the Golgi apparatus. This inability to fuse was not the result of the lack of Sed5p, because vesicles specifically depleted of Sed5p generated by antibody inhibition targeted and fused normally. We propose that the A-site of Sec24p is a multipurpose cargo-binding site that must recognize additional unidentified cargo proteins, at least one of which is essential at a late stage of vesicle fusion.     

Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif

Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion. However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly. We now show that Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates. In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain. These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception.     

Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions

Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE-SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.     

Cysteine-disulfide cross-linking to monitor SNARE complex assembly during endoplasmic reticulum-Golgi transport

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro-alpha factor. This data suggests that Ca(2+) is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.     

SM-protein-controlled ER-associated degradation discriminates between different SNAREs

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a specialized activity of the ubiquitin-proteasome system that is involved in clearing the ER of aberrant proteins and regulating the levels of specific ER-resident proteins. Here we show that the yeast ER-SNARE Ufe1, a syntaxin (Qa-SNARE) involved in ER membrane fusion and retrograde transport from the Golgi to the ER, is prone to degradation by an ERAD-like mechanism. Notably, Ufe1 is protected against degradation through binding to Sly1, a known SNARE regulator of the Sec1-Munc18 (SM) protein family. This mechanism is specific for Ufe1, as the stability of another Sly1 partner, the Golgi Qa-SNARE Sed5, is not influenced by Sly1 interaction. Thus, our findings identify Sly1 as a discriminating regulator of SNARE levels and indicate that Sly1-controlled ERAD might regulate the balance between different Qa-SNARE proteins.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

Evidence for regulation of ER/Golgi SNARE complex formation by hsc70 chaperones

SNARE proteins control intracellular membrane fusion through formation of membrane-bridging helix bundles of amphipathic SNARE motifs. Repetitive cycles of membrane fusion likely involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, little is known about conformational regulation of SNAREs by other proteins. Here we demonstrate that hsc70 chaperones stimulate in vitro SNARE complex formation among the ER/Golgi SNAREs syntaxin 5, membrin, rbetl and sec22b, under conditions in which assembly is normally inhibited. Thus, molecular chaperones can render the SNARE motif more competent for assembly. Partially purified hsc70 fractions from brain cytosol had higher specific activities than fully purified hsc70, suggesting the involvement of unidentified cofactors. Using chemical crosslinking of cells followed by immunoprecipitation, we found that hsc70 was associated with ER/Golgi SNAREs in vivo. Consistent with a modulatory role for hsc70 in transport, we found that excess hsc70 specifically inhibited ER-to-Golgi transport in permeabilized cells.     

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.     

p115–SNARE Interactions: A Dynamic Cycle of p115 Binding Monomeric SNARE Motifs and Releasing Assembled Bundles

Tethering factors regulate the targeting of membrane‐enclosed vesicles under the control of Rab GTPases. p115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, the mechanism of action of p115 is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that function of p115 is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from coat protein II (COPII) vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at prefusion sites and is subsequently ejected as SNARE complex formation and fusion proceed.      

The yeast Batten disease orthologue Btn1 controls endosome-Golgi retrograde transport via SNARE assembly

The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.     

The Dynamics of Golgi Protein Traffic Visualized in Living Yeast Cells

We describe for the first time the visualization of Golgi membranes in living yeast cells, using green fluorescent protein (GFP) chimeras. Late and early Golgi markers are present in distinct sets of scattered, moving cisternae. The immediate effects of temperature-sensitive mutations on the distribution of these markers give clues to the transport processes occurring. We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and sed5 cells, but not in sec14 cells. This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early cisternae containing the target SNARE Sed5p. Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying that it cycles through the ER. Electron microscopy shows that Golgi membranes vesiculate in sec18 cells within 10 min of a temperature shift. These results emphasize the dynamic nature of Golgi cisternae and satisfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, either within the Golgi complex or from there to the ER, as anterograde cargo advances.