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THE DSL1 TETHERING COMPLEX IS A RESIDENT ER COMPLEX, WHICH INTERACTS WITH FIVE SNARES - IMPLICATIONS FOR FUSION AND FUSION REGULATION

Christoph T.A. Meiringer1,*, Ralf Rethmeier1,*, Kathrin Auffarth1, Joshua Wilson2, Angela Perz1, Charles Barlowe2, Hans Dieter Schmitt3, and Christian Ungermann1

University of Osnabrück1, Department of Biology/Chemistry, Biochemistry Section, Barbarastrasse 13, 49076 Osnabrück, Germany

Department of Biochemistry2, Dartmouth Medical, School, Hanover, NH 03755, USA Department of Neurobiology3, Max Planck Institute for Biophysical Chemistry, D-37070 Göttingen,

Germany Running head: Interactions between SNAREs and the Dsl1 complex

Address correspondence to: Christian Ungermann, Department of Biology/Chemistry, Biochemistry Section, Barbarastrasse 13, 49076 Osnabrück, Germany; Fax: +49-541-969-2884; E-mail: cu@uos.de

Characters (without spaces): 27,281

Keywords: Dsl1 complex, Ykt6, tethering, retrograde transport, SNARE, vesicle

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 multi-subunit 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 Golgi-derived vesicles at the ER 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 unaffected. Reconstitution approaches reveal that the Dsl1 complex contains several interfaces for SNAREs, and in

http://www.jbc.org/cgi/doi/10.1074/jbc.M110.215327The latest version is at JBC Papers in Press. Published on May 6, 2011 as Manuscript M110.215327

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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vivo studies suggest that subunits of the Dsl1 complex and the Q-SNAREs are ER-resident proteins. Our data support a model of tethering via coat recognition, followed by SNARE assembly and fusion.

Experimental Procedures

Yeast strains and plasmid construction -

Yeast strains used in this study are listed in Table S1. These were either generated by homologous recombination of PCR-amplified fragments or by transformation with plasmids (see below). For yeast two-hybrid analysis full-length or truncated ORFs were cloned into both pACT2 (Clonetech) and pFBT9 (15) plasmids and co-transformed into yeast strain PJ69-4A (listed in Table S2). Transformants were selected on SDC-LEU-TRP plates and four clones of each tested interaction were re-streaked to QDO plates (SDC-LEU-TRP-HIS-ADE). Growth was assayed after 4-7 days. Plasmids for purification of recombinant proteins were cloned either into pGEX-2TK (GE Healthcare), pETHIS (14) or pET32c(-Trx) (modified from pET32c(+), Novagen, by removal of the thioredoxin tag) and are listed in Table S3. The SNAREs were cloned without their transmembrane domain. PCR amplification was performed using Pfu polymerase (Fermentas GmbH) and all Y2H-plasmids were sequenced (GATC Biotech AG). Restriction digest and cloning were performed according to manufacturer (Fermentas GmbH).

Microscopy - Yeast cells expressing either Tip20-mGFP, Dsl1-GFP or Dsl3-mGFP were grown to early log-phase, harvested, washed once with PBS and mounted on object slides. Visualization was performed on a fluorescence microscope (Leica DM5500 B; Leica Microsystems GmbH) equipped with a GFP-filter (Exc. D480/30, Em. D535/40, Beamsplitter 505dclp; Chroma Technology), captured with a digital camera (Spot Pursuit-4MP; Diagnostic Instruments, Inc.) and processed using Metamorph (Molecular Devices) and Autoquant X (Media Cybernetics). Strains bearing a temperature-sensitive allele were analysed both after growth at 23°C and after a temperature shift to 37°C for 30 min. For Dsl1 and Ypt1 depletion experiments strains were grown in YPG, washed once in PBS and then grown in YPD for nine hours.

Tandem Affinity Purification - TAP-tag protein tandem affinity purification was performed as described in (16,17) using the

following buffer: 50 mM HEPES/KOH, pH 7.4, 150 mM NaCl, 0.15% NP-40 (Igepal CA-630; Sigma-Aldrich), and 1.5 mM MgCl2. The buffer was supplemented with 1 mM PMSF, 1 mM DTT, and 1x Protease Inhibitor Mix FY (Serva). For washing of IgG-Sepharose (GE Healthcare), the buffer was supplemented with 0.5 mM DTT.

Additional Methods are provided in the Supplemental materials.

RESULTS

Isolation of the Dsl1 complex identifies the

R-SNARE Ykt6 - The SNARE Ykt6 is distributed between cytosol and membranes. To identify a potential receptor of Ykt6, we tagged Ykt6 with GFP in a loop contained within its N-terminal longin domain and isolated the protein using antibodies against GFP coupled to Protein A Sepharose. Proteins that co-eluted with Ykt6-GFP were identified by mass spectrometry (Figure 1A). Besides known interaction partners (Sed5, Sec17 and Sly1), we were able to identify Dsl1, Dsl3 and Tip20 in the eluate. All three proteins belong to the Dsl1 complex at the ER, localize similarly to punctate structures at the cortical and perinuclear ER (Figure 1B), and are equally abundant (Figure 1C), as reported (2).

To confirm the interaction between the Dsl1 complex and Ykt6, we tagged Dsl1 and Dsl3 with a C-terminal TAP-tag and performed a tandem affinity purification via IgG Sepharose and CaM-beads (Figure 1D, S1A). Using mass spectrometry, we identified not only the three Dsl1 complex subunits, but also all of the components of the ER SNARE complex Ufe1, Use1 and Sec20. This stable complex of tethers and SNAREs was previously reported, but lacked the appropriate R-SNARE (2). When we subjected the eluate of the IgG beads to gel filtration, we recovered the Dsl1 complex together with the previously identified Q-SNAREs and Ykt6 in a high molecular weight complex of approx. 700 kDa in fractions 11, 12 (Figure 1D, S1A), and a subcomplex of Dsl1 and Dsl3 in fractions 13 and 14 (Figure 1D), in agreement with recent structural work (3).

Sec22 and Ykt6 interaction with the Dsl1 complex differs - Sec22 was previously described to be the R-/v-SNARE in transport between the Golgi and ER (11,18,19), though we found Ykt6 (Figure 1). However, we detected both Sec22 and Ykt6 in association with Dsl3-TAP and the other subunits of the Dsl1 complex on Western blots (Figure S1B), whereas control

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beads did not precipitate any SNARE (Figure 2E). We therefore wondered if we could detect differences in the composition of the Dsl1 tether complex with SNAREs and thus determine the precise interactions. We tested different ts mutants of the Dsl proteins, Dsl1 and Tip20, the SNARE Sec20 and Sec18, the ATPase required for SNARE complex disassembly. The composition of the Dsl1 complex and its interaction with SNAREs were unchanged in the sec18-1 mutant, which is blocked in SNARE disassembly at the restrictive temperature (Figure 2A, lane 14, Figure S1B). In contrast, mutants in dsl1 (lane 2-6), or tip20 (lane 9-12) strongly affected the stability of the Dsl1 complex (Figure 2A). This indicates that the interaction of the Dsl1 complex with all five SNAREs is affected by Dsl1 complex mutations, but not by alterations in the SNARE chaperone Sec18.

To unravel if the two R-SNAREs compete for the same binding site on the complex, we overexpressed either Sec22 or Ykt6, and purified the Dsl1 complex via Dsl3. Overexpression of Ykt6 did not have any significant effect on Sec22 binding to the Dsl1 complex, whereas Sec22 overexpression completely abolishes binding of Ykt6 (Figure 2B, lane 3 vs. 4). We then asked whether the competition of Ykt6 and Sec22 may be enhanced if the turn-over of the SNARE complex was impaired. Indeed, overexpression of Ykt6 in the sec18-1 mutant was sufficient to displace Sec22 from the Dsl1 complex (Figure 2C), indicating that Ykt6 can compete with Sec22 under these conditions. Moreover, these cells grew more slowly at higher temperatures than cells with the sec18-1 allele alone (Figure 2D). It was previously demonstrated that Ykt6 functionally replaces Sec22 in transport between the Golgi and the ER (13). We therefore reasoned that Ykt6 might be enriched in the Dsl1 complex, if Sec22 is lacking. Contrary to our assumption, the pull-down of Dsl3-TAP in a sec22∆ strain did not show a significant accumulation of Ykt6 in the Dsl1 complex (Figure 2B, lane 2). We conclude that association of Ykt6 with the Dsl1 complex occurs upstream of Sec22 binding and the latter prevents Ykt6 from reentering the complex.

We then asked if the interaction of Sec22 and Ykt6 with the Dsl1 complex is altered by the addition of purified Sec18, which can disassemble SNARE complexes (20). When ER-enriched membranes were incubated with Sec18 and ATP, only Ykt6 was selectively displaced from Sec20 or Dsl3, whereas the remaining

complex stayed intact (Figure 2E, Figure S1C, lane 4). Our data suggest that Ykt6 occupies a Sec18-sensitive binding site on the assembled SNARE-Dsl1 complex.

The Dsl1 complex binds the Q-SNAREs Sec20 and Use1 via their N-terminal domains - To map the interactions between components of the Dsl1 complex and SNAREs, we employed yeast-two hybrid analysis. Interestingly, several subunits like Use1 and Dsl1 showed multiple interactions, suggesting that they occur in the context of the partially assembled Q-SNARE-Dsl1 complex (Figure S1D). We therefore focused on the N-terminal (NT) and the SNARE domains (SD) of each Q-SNARE (Figure 3A). Our data indicate that the Dsl1 complex recognizes the N-terminal domains of the SNAREs Use1 (1-141) and Sec20 (1-196) via its subunits Dsl3 and Tip20, in agreement with Hughson and colleagues (21) (Figure 3A, B). For Tip20, we mapped the binding site for Sec20 to residues 377-611 (Figure 3C, D), C-terminal of the proposed binding site by Ren et al. (21). Whereas Tripathi et al. mapped the interaction site of Tip20 to Dsl1 to the first 41 residues (3), an internal deletion of residues 80-110 also abolished the binding to Dsl1, which may be due to changes in secondary structure. Alternatively, the binding site between Tip20 and Dsl1 includes additional Tip20 segments.

To test the interactions directly, we purified the components of the Dsl1 complex and reconstituted the complex assembly. We detected direct binding of Dsl1 to Dsl3 and Tip20, but not for Tip20 to Dsl3 (Figure 3E). This is in agreement with previously published structural findings (3,21). By gel-filtration, we showed that monomeric Tip20 (Figure 3F) assembled into the large Dsl1 complex (fractions 45-50), if incubated with Dsl1 and Dsl3 (Figure 3G), and some large complex in the void volume. When the concentrated Dsl1 complex was incubated with an excess of either Sec20 or Sec22, only Sec20 bound to the Dsl1 complex, presumably via Tip20 (Figure 3H). The reconstitution of the Dsl1 complex with all SNAREs, including the R-SNARE, was very inefficient (not shown), and the interaction may occur only transiently in vivo. Our data reveal that the Dsl1 complex has binding sites for the N-terminal domains of Sec20 and Use1, and plays a role in the assembly of the SNARE complex, consistent with our initial observation (Figure 1).

Evidence for an ER-resident Dsl1 complex - If Sec22 were the retrograde v-SNARE, it

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appears unlikely that it is replaced by acylated Ykt6, which lacks the transmembrane domain that is required to drive fusion of the lipid bilayers. Moreover, the interaction of Dsl1 and COPI suggested that Dsl1 could be recruited to retrograde vesicles. We therefore took advantage of a COPII budding assay (22) to monitor the incorporation of selected subunits of the Dsl1 complex and SNAREs (Figure 4A). Vesicles generated in the presence of COPII components contained Sec22 and the cargo receptor Erv26, but lacked Dsl1, Tip20 and Ufe1, consistent with the predominant ER-localization of the Dsl1 complex in vivo (Figure 1B). As we lacked an antibody to Sec20, we turned to an in vivo assay, using functional GFP-tagged Sec20 (Figure 4B). Sec20 has been previously reported to contain a luminal HDEL motif, which might be responsible of its ER localization (23). However, neither sec22 deletion, Ykt6 overexpression, Ypt1 depletion, mutations in the δ-COP (ret2-1), nor alterations in Dsl3 functionality affected the steady-state localization of Sec20 to the ER (Figure 4C, D). In the sec18-1 mutant, we observed accumulations of GFP-Sec20, which increased in size at the restrictive temperature (Figure 4D). These dots colocalized with the ER marker Sec63 (Figure S2D). They might be the result of additional Ykt6 associated with the (Dsl1-)SNARE complex, which may impair transport between ER and Golgi, and subsequently growth (Figure 2D). Our data therefore suggest that it is unlikely that Sec20 functions as a v-SNARE on retrograde vesicles.

Furthermore, depletion of Ypt1 or Dsl1 - the latter one causes a massive accumulation of COPI vesicles (8) - did not affect Dsl3 localization to the ER (Figure 4E). Upon depletion of Dsl1, Sec20 and Dsl3 showed colocalization with the ER marker Sec63, but not with the Golgi marker Mnn9 (Figure S2A, B). Also Dsl1 itself, which does not bind directly to a SNARE or any other ER transmembrane protein, seem to be stably localized at the ER. In ret2-1 cells or a ts mutant of the ER to Golgi v-SNARE bos1 (bos1-1), Dsl1 is found at the ER (Figure 4F), similar to our observations upon Ypt1 depletion (data not shown). Our data are therefore consistent with a stable ER-resident Dsl1 complex, which is kept in place by binding ER-resident Q-SNAREs.

DISCUSSION

Fusion of Golgi-derived vesicles with the ER requires a close cooperation of the Dsl1 complex (2) with the SNAREs Ufe1, Use1, Sec20 and Sec22 (12,19). Previous purification of the Dsl1 complex did not yield any R-SNARE, but only the Q-SNAREs Use1, Ufe1 and Sec20 (2). However, Use1 interacts with Sec20, Ufe1, and the R-SNAREs Sec22 and Ykt6 (19). We now demonstrate that Sec22 and Ykt6 are found in substoichiometric amounts in association with the isolated Dsl1 complex, suggesting that Ykt6 may regulate vesicle fusion by binding not only the Q-SNAREs, but also the SNARE-Dsl1 complex. We could not identify a binding site between any Dsl1 complex subunit and Ufe1 or Sec22, indicating that both bind to the Dsl1 complex via the SNAREs Sec20 and Use1 (11), which bind Tip20 and Dsl3 via their N-terminal domains (Figure 3A, B)(21). Our data suggest that Sec22 is indeed the missing R-SNARE, as expected from previous studies (12,19). Overexpression of Sec22 completely removes Ykt6 from its association with the Dsl1 complex, whereas Ykt6 could not do so in reverse. A similar association of Ykt6 with the Dsl1-SNARE complex was recently observed by Spang and colleagues (37). In addition, Sec18 addition displaced Ykt6, but not Sec22 or the Q-SNAREs, from the Dsl1 complex. It is therefore possible that Ykt6 acts as an acceptor for the preferred SNARE Sec22 at the ER. Our data is consistent with the view that Sec22 is the v-SNARE in vivo, whereas the Q-SNAREs seem to be resident proteins of the ER.

While our manuscript was in preparation, two studies provided insight into the structure of the Dsl1 complex (3,21). Dsl1 and Tip20 resemble known structures of the Exocyst complex (3). In agreement with our results, Hughson and coworkers demonstrate that Use1 and Sec20 bind via their N-termini to Dsl3 and Tip20, respectively (21). Our data indicate that Ufe1 requires Sec20 for binding, whereas Sec22 only binds if all other SNAREs are present. This observation is consistent with our initial isolation of Sec22 and Ykt6 with the Dsl1 complex from yeast (Figure 1, 2). The purified Dsl1 complex is, however, very inefficient in promoting SNARE assembly (3), and it has not been tested, if the slight increase in complex formation correlates with increased fusion.

The Dsl1 complex is a critical factor involved in COPI vesicle recognition at the ER (8). Its subunit Dsl1 binds directly to heterodimeric complex of ε-COP and the C-terminus of α-COP (24), presumably to tether

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the COPI vesicles to the ER and promote uncoating. Our data agree with such a combined function, and suggest that the ER resident Dsl1 complex then promotes assembly of Sec22 with the Dsl1 complex-bound Q-SNAREs.

At the Dsl1 complex, Ykt6 may occupy a binding site to facilitate the association of Sec22 with the Q-SNAREs. Indeed, Sec22 can displace Ykt6, and the reverse is also possible if Sec18 is impaired. Potentially, Ykt6 orients the Dsl1-Q-SNARE complex in such a way that is predominantly competent to bind Sec22 in trans. If this is the case then Ykt6 would have a more active role in ensuring proper assembly. We consider it unlikely that Ykt6 functions as a v-SNARE on Golgi-derived vesicles, because its lipid anchor does not support membrane fusion (25). However, we cannot exclude a function of Ykt6 on COPI vesicles if Sec22 is lacking. It is also possible that in sec22∆ cells, a small portion of Sec20, Ufe1 or Use1 are recycled via the Golgi back to the ER and thus act as a v-SNARE, though we did not observe incorporation of Ufe1 into COPII coated vesicles, or relocalization of Sec20 into Golgi-like structures, except some dot-like structures in sec18-1 mutants (Figure 4C, D). It should be noted that more Ykt6 enters COPII coated vesicles in sec22∆ cells (13), suggesting that Ykt6 may compensate for some of Sec22’s functions. Alternatively, the Golgi SNARE Bet1, which interacts with Ufe1 and Use1 in vitro (37), may function in this process.

For the mammalian homolog of Dsl1, ZW10, it has been proposed that the protein is cycling between the ER and the Golgi (26). This model is based on the observations that in primate cells ZW10 localizes at the ER while in rodent cells it is found at the Golgi (27-29). Our data on the yeast Dsl1 complex support a model, in which the entire tethering complex resides on one organelle and recruits vesicles via their coat (30). Transport vesicles should then maintain their coat to ensure the recognition at the target organelle. Our interaction studies confirm that the Dsl1 complex binds the N-terminal domains of Use1 and Sec20, though it is possible that the complex has additional membrane binding sites. This would position the complex such that it directly couples tethering of COPI vesicles and SNARE-mediated fusion, by bringing together the SNARE on the vesicle with the assembled t-SNARE complex on the ER membrane (Figure 4G). Interestingly, such a scenario is reminiscent of the proposed interaction of the HOPS complex with the AP-3 coat. Here, the AP-3 subunit Apl5 binds the HOPS subunit Vps41 (31-33), which is regulated by phosphorylation (34). HOPS might get stabilized similarly by SNAREs on the vacuole (35), and could then tether AP-3 vesicles by binding the coat. We therefore postulate that the tether-coat interaction might be of general importance to drive fusion reactions.

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FOOTNOTES

*C.M. and R.R. contributed equally to this study; C.M.’s present address: Roche Diagnostics

GmbH, Nonnenwald 2, 82377 Penzberg, Germany. We thank Fred Hughson for the Dsl3 expression plasmid, and all members of the Ungermann lab for fruitful discussions. This work was supported by the DFG (SFB 431, 944, and UN111/4-2), and the Hans-Mühlenhoff foundation (to C.U.).

FIGURE LEGENDS

Fig. 1. Ykt6 interacts with the Dsl1 complex. A. Components of the Dsl1 complex are co-

purified with Ykt6-GFP. Ten thousand OD-units of yeast strain overexpressing Ykt6-GFP (GAL1-YKT6-GFP) were lysed and processed as described in Methods, and subjected to immunoprecipitation using affinity purified GFP antibodies coupled to Protein A Sepharose. Eluates were applied to 4-12% SDS-gels and prominent bands analyzed by mass spectrometry after colloidal Coomassie staining. B&C. In vivo localization of Dsl1 complex subunits. C-terminally tagged Dsl1 complex subunits were

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analyzed by fluorescence microscopy (B; size bar indicates 10µm). Subcellular fractionation of yeast cell lysates was done with TAP-tagged Dsl1, Dsl3 or Tip20. Proteins from membrane fraction were analyzed by SDS-PAGE and Western blotting using antibodies against the calmodulin binding peptide of the tandem affinity purification (TAP) tag or Vac8 (C). D. Ykt6 is found in association with the Dsl1 complex. Dsl1-TAP was purified either by tandem affinity purification or on IgG-Sepharose alone and subjected to gel filtration on a superose-6 column as described (36). Aliquots of the resulting fractions were analyzed on SDS-PAGE (Coomassie stained). Fraction numbers are indicated below the gel. Prominent bands of the tandem affinity purification were cut for identification in mass spectrometry. Cbp, calmodulin binding peptide.

Fig. 2. Differential binding of R-SNAREs to the Dsl1 complex. A. Analysis of ts-strains for the

interaction between SNAREs and the Dsl1 complex. Cells were grown overnight at 23°C and heat shocked at 37°C for 2h. Lysates of 10,000 OD600 units of cells were pepared as described in methods for TAP. After purification and TEV elution, 10% of the TEV-eluate was loaded on a 4-12% SDS-PAGE gel, and proteins were analyzed by Western blotting using the indicated antibodies. B. Overexpression of Sec22 displaces Ykt6 from the Dsl1 complex. Control cells (wt), sec22Δ cells, or cells overexpressing Ykt6 (GAL1-YKT6) or Sec22 (GAL1-SEC22) were grown over night in YPG. Dsl3-TAP was purified from 1500 OD600 units via IgG sepharose and TEV cleavage. Eluates were analyzed as in (A). L, load; FT, flowthrough; cbp, calmodulin binding peptide. wt, wild-type. C. In sec18-1 background, overexpression of Ykt6 displaces Sec22 from the Dsl1 complex. Cells were grown in YPG at 23°C and shifted to 37°C for 3 hrs. Sec20-TAP pull down was made from indicated strains as in (B). Note that Sec22 is not well visible in the Ykt6 overexpression lane to the Ykt6 signal in the western blot. D. Ykt6 overexpression in sec18-1 strain enhances the temperature sensitive phenotype. Dilution series from 0.25 to 0.000025 OD600 of indicated strains were grown at 23, 30, and 37°C and photographed. E. Sec18 displaces Ykt6 from the Dsl1 complex. A membrane fraction (P10) was prepared from 3000 OD600 units of the indicated cells as described in Methods, and incubated in reaction buffer with or without Sec18 and ATP. Membranes were re-isolated for 10 min at 20,000g at 4°C, resuspended in lysis buffer and the TAP-tagged proteins were isolated as before. TEV-eluted proteins analyzed as in (A). wt, wild-type; S20T, Sec20-TAP; D3T, Dsl3-TAP.

Fig. 3. Subunit interactions between Dsl1 complex and SNARE proteins. A&B. Q-SNARE

domain interaction with Dsl1, Dsl3 and Tip20. A. Interactions between fragments of the N-terminal regulatory domain (NT) and SNARE domain (SD) of Ufe1, Use1 and Sec20 and full-length Dsl1, Dsl3 and Tip20 were analyzed by yeast two hybrid. B. Pull down with GST-Tip20 against cytosolic domains of Sec20. Full length Tip20 bound to GSH beads was incubated with the cytosolic part of Sec20 (Cy, w/o Trx-tag), only N-terminus (NT) or SNARE domain (SD, both with Trx-tag). GSH beads were eluted by boiling in Laemmli buffer and the samples were analyzed by SDS-PAGE followed by Coomassie staining (Coom., upper panel). In addition, a Western Blot was decorated with His-tag antibody (WB, lower panel). C&D. Mapping of Tip20 interactions. C. Interactions between truncations of Tip20 and full length Dsl1 and Sec20 were analyzed by yeast two hybrid. D. Graphic depiction of (C). E. Reconstitution of the Dsl1 complex. Purified Dsl1 and Dsl3 were added to GSH beads carrying GST or GST-Tip20 and the pull-down assay was performed as described in Methods and analyzed by SDS-PAGE and Western blotting. F. Migration of proteins of the Dsl1 complex and indicated SNAREs on gel-filtration. The indicated purified proteins were analyzed by gelfiltration on a Sephacryl S-300 HR column (GE Healthcare). One ml fractions were collected, TCA-precipitated and analyzed by SDS-PAGE and Coomassie staining. Arrows indicate the single gels. G. Reconstitution of the Dsl1 complex. Equimolar amounts of the Dsl3, Dsl1 and Tip20 lacking the GST were incubated at 4°C and analyzed by gelfiltration as in (F). H. Reconstitution of Dsl1 complex interaction with Sec22 and Sec20. Fractions between 45 and 50 ml of reconstituted Dsl1 complex as in (G) were concentrated, incubated with 2-fold molar excess of Sec20 and Sec22 and analyzed by gel filtration. One ml fractions were collected and analyzed as in (F).

Fig. 4. Analysis of Q-SNARE and Dsl1 complex cycling between ER and Golgi. A. Dsl1, Tip20

and Ufe1 are not incorporated in COPII vesicles. Purified ER membranes were incubated with COPII subunits. Vesicles were then isolated, and proteins analyzed by SDS-PAGE and Western blotting using antibodies against the indicated proteins. 10% of a total reaction (T) was compared to budded

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8

vesicles produced in the absence (-) or presence (+) of COPII proteins. B. GFP-Sec20 is functional. Cells carrying a sec20-1 ts mutant or GFP-Sec20 were serially diluted, spotted on YPD plates, and incubated at the indicated temperatures. C. Neither Ykt6 overexpression, SEC22 deletion nor Ypt1 depletion affect Sec20 localization. D. Localization of GFP-Sec20 in selected mutants. E. Localization of Dsl3-GFP at the ER is not disturbed by depletion of Dsl1 or Ypt1. Western blots showing Ypt1 and Dsl1 depletion are in Figures S1E and S2C. F. Dsl1-GFP localizes at the ER in ts mutants defective in ER to Golgi (bos1-1) or Golgi to ER (ret2-1) transport. For (C) – (F) microscopy of GFP constructs was done as described in Methods. Size bar indicates 10 µm. G. Model of the Dsl1 complex assembled with the three Q-SNAREs Sec20, Ufe1 and Use1 and docked COPI vesicle. Whereas Sec20 and Use1 bind directly to Tip20 and Dsl3 via their N-terminal domains, Ufe1 does not interact with the Dsl1 complex as well as the R-SNAREs Sec22 and Ykt6. Ykt6 is lacking a transmembrane domain and associates to membranes by a farnesyl and a palmitoyl anchors.

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Sly1, Tip20SSA2 (Hs)

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Figure 1, Meiringer et al.

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-

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+n.d.

-n.d.

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Figure 3, Meiringer et al.

1 701221

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SUPPLEMENTAL MATERIALS (Meiringer et al.)

Content: Supplemental Methods and Legends Table S1: Yeast strains used in this study Table S2: Expression plasmids used for GST and His6-purification from E. coli. Table S3: Plasmids used in yeast two-hybrid analysis Figure S1: Interactions of Ykt6 with the Dsl1 complex Figure S2. Localization of Dsl3 and Sec20 under Dsl1 depletion condition.

Supplemental Methods

IgG pull-down assay from membrane fractions - Cells were grown to log phase (OD600=1.5-2.5) and 1500 OD-units were harvested. The cell pellet was resuspended in 100 mM Tris/HCl, pH 9.4, 10 mM DTT and incubated at 30°C for 10 minutes. Afterwards, cells were retrieved by centrifugation, resuspended in SB-Buffer (0.2x YPD, 600 mM Sorbitol, 50 mM KPi, pH 7.4) and incubated with 2 ml of lyticase at 30°C for 20 minutes. Spheroblasts were recovered by centrifugation and lysed in PEKS-Buffer (10 mM Pipes/KOH pH 6.8, 2 mM EDTA, 50 mM KAc, 200 mM sorbitol, supplemented with Protease Inhibitor Mix FY (Serva), 1 mM PMSF and 1 mM DTT) and 0.4 mg/ml DEAE-Dextran. Unbroken cells and nuclei were removed by centrifugation at 300 g for 10 minutes at 4°C, and the cleared lysate was centrifuged for 15 minutes at 10,000 g. The resulting pellet (P10) was solubilized in L-Buffer (50 mM HEPES/KOH, pH 7.5, 150 mM NaCl, 0.15% Igepal C-630, 1.5 mM MgCl2) supplemented with FY, 1 mM PMSF and 1 mM DTT. After clearing of the lysate at 10,000 g for 15 minutes, it was incubated for 1 hour with IgG-Sepharose (GE Healthcare). IgG-Sepharose beads were then transferred to MoBiCols (MoBiTec GmbH) and washed with 15 ml of L-Buffer without protease inhibitors and only 0.5 mM DTT. Beads were then resuspended in 250 µl of L-Buffer as before and TAP-tagged proteins eluted by incubation with 2.5 µl of TEV-protease either at 4°C over night or at 16°C for 2 hours. The eluate was collected by centrifugation, and TCA-precipitated at 13% TCA final concentration, washed with ice-cold acetone and resuspended in SDS-sample buffer (Invitrogen GmbH). Proteins were separated on 4-12% Tris-Glycine SDS-gels (Invitrogen GmbH), blotted to nitrocellulose membranes and decorated using the described antibodies.

SNARE complex disassembly by Sec18 - Yeast P10-fractions were obtained as described for IgG pull-down assay from membrane fractions. These were resuspended in 0% Ficoll and 1 mg was incubated in a release reaction containing ATP and Sec18 in fusion reaction buffer as described (1). Membranes were re-isolated by centrifugation (13,000g at 4°C) and either processed as described above or subjected after lysis to glycerol gradient analysis as described (2).

Recombinant Proteins - Recombinant Dsl1 complex proteins from pGEX-2TK were prepared from Escherichia coli BL21 Rosetta as previously described (3). SNARE proteins without transmembrane domain were expressed from pET32c(-Trx). In brief, cells were grown to OD600=0.4-0.6 and induced with 0.4 mM IPTG over night at 16°C. Cells were harvested, resuspended in Buffer I (50 mM NaH2PO4, pH 8.0, 500 mM NaCl, 0.1% IGEPAL C-630, 10% Glycerol) supplemented with 12 mM 2-mercaptoethanol, 1x PIC and 2 mM PMSF and lysed by sonication or Microfluidizer Processor (Microfluidics). Cleared lysate was incubated with Ni2+-NTA (Qiagen GmbH) or GSH-Sepharose (GE Healthcare) and after washing, bound proteins were either eluted with Buffer I containing 250 mM imidazole, or cleaved with thrombin followed by cleanup with benzamidine-agarose (Sigma Aldrich) for pET32c(-Trx) and pGEX-2TK constructs, respectively. Buffer-exchange was performed on PD10 columns (GE Healthcare) to final reaction buffer for GST pull down and gel filtration experiments.

GST pull down - Ten µg each of indicated proteins purified from E. coli in Buffer II (50 mM NaH2PO4, pH 8.0, 150 mM NaCl, 0.1% IGEPAL C-630, 5% Glycerol) were incubated in a total reaction volume of 100 µl on 10 µl GSH-Sepharose (GE Healthcare Life Science) over night at 4°C.

GSH-Sepharose with bound proteins were recovered by centrifugation and washed three times each with 800 µl of Buffer II. Proteins were eluted by addition of 15 µl of SDS-sample buffer and boiling for 5 minutes.

Gel-Filtration - For reconstitution of Dsl1 complex, 3 µM of recombinantly expressed Dsl1, Dsl3 and Tip20 were incubated in 1 ml reaction volume for 1hr at 4°C, and then centrifuged for 20 min at 20,000 g. Gel-filtration was done on a Sephacryl S-300 HR (GE Healthcare) at 4°C in the same buffer like the incubation before (50 mM Tris/Cl, pH 8.0, 150 mM NaCl, 2 mM DTT) with a flow rate of 0.2 ml/min. One ml fractions were collected and TCA precipitated, and resuspended in SDS-sample buffer. For the reconstitution of the Dsl1 complex together with the SNAREs, fractions of Dsl1 complex reconstitution (45-50 ml) were pooled and concentrated on Vivaspin 2 MWCO 50,000 (GE Healthcare) to 500 µl. Afterwards, the SNAREs were added to the complex, followed by incubation and gel-filtration as before. Samples were analyzed on 12% SDS-PAGE gels.

Subcellular Fractionation - Fractionation of yeast membranes was carried out as described (4). Yeast genetically modified to carry a TAP tag at the C-terminus of either Dsl1, Dsl3 or Tip20 were osmotically lysed and membranes fractionated by differential centrifugation. After a clearing run at 300 g, the supernatant was sequentially spun at 10,000 and 100,000g. The S100 supernatant after the last spin was TCA-precipitated, all pellets were resuspended in SDS-sample buffer, and examined by SDS-PAGE and Western blotting using secondary antibodies coupled with either Alexa 680 or IRDye 800.

SUPPLEMENTAL FIGURES

Fig. S1. Interactions of Ykt6 with the Dsl1 complex. A. Ykt6 is found in association with the Dsl1 complex. Dsl3-TAP was purified either by tandem affinity purification or on IgG-Sepharose alone and subjected to gel filtration on a superose-6 column as described [5]. Aliquots of the resulting fractions were analyzed on SDS-PAGE (Coomassie stained). Fraction numbers are indicated below the gels. Prominent bands of the tandem affinity purification were cut for identification in mass spectrometry. Cbp, calmodulin binding peptide. B. Interaction of the Dsl1 complex with Sec22 and Ykt6. Cells carrying TAP-tagged Dsl3 (D3T) with wild-type or temperature sensitive Sec18 (D3T sec18) were grown at 23°C over night, and heat shocked for 1 hr at 37°C, where indicated. Cells were lysed, and Dsl3 was purified via IgG beads and TEV cleavage from the lysate. Eluates were examined by SDS-PAGE and Western blotting, using antibodies against the indicated proteins. C. Sizing of the Dsl1 complex after Sec18 disassembly. Release reactions were prepared as in Figure 2C with membrane fractions from yeast strain expressing Dsl3-TAP. The re-isolated membranes were lysed and loaded on a 10-30% glycerol gradient and centrifuged for 18 hrs at 250,000 g. One ml fractions were collected, TCA precipitated and analyzed by SDS-PAGE and Western Blot. D. Interaction of full-length Dsl1 complex proteins and SNAREs in yeast two-hybrid assay. E. Depletion of Ypt1. Cells were grown in YPG, washed in PBS and then diluted into YPD and incubated for 9hrs. At indicated time points 1 OD600 was spun down, boild in Lämmli buffer and SDS-PAGE was done followed by Western Blotting. Blots were decorated against indicated proteins.

Fig. S2. Localization of Dsl3 and Sec20 under Dsl1 depletion condition. A&B. Depletion of

Dsl1 does not disturb the colocalization of Dsl3 (A) or Sec20 (B) with Sec63 at the ER. Colocalization with the Golgi marker protein Mnn9 cannot be observed. Cells were treated like described in methods. Size bar indicates 10 µm. C. Depletion of Dsl1. Cells were grown in YPG, washed in PBS and then diluted into YPD and incubated for 9hrs. At indicated time points 1 OD600 was spun down, boiled in Laemmli buffer and SDS-PAGE was done followed by Western Blotting. The blot was decorated against indicated proteins. D. Colocalization of Sec20-GFP in sec18-1 mutant with Sec63. RFP-tagged Sec63 was co-expressed in the GFP-Sec20 background and analyzed by fluorescence microscopy. Sec20 colocalizes with the ER marker Sec63 in punctuated structures at 23° and more so at 37°C. Size bar indicates 10 µm.

SUPPLEMENTAL TABLES

Table S1. Yeast strains used in this study. Strain Genotype Source/Reference

BY4741 MATa ura3∆0 leu2∆0 his3∆0 met15∆0 EuroscarfTM BY4727 MATalpha his3∆200 leu2∆0 lys2∆0 met15∆0 trp1∆63 ura3∆0 EuroscarfTM

PJ69-4A MATa trp1-901 leu2-3-112 ura3-52, his3-200 gal4∆ gal80∆ GAL2-ADE2 LYS2::GAL1-HIS3 met2::GAL7-lacZ (6)

YSC1178-7502517 MATa DSL1-TAP::HIS3MX ura3Δ0 leu2Δ0 his3Δ met15Δ0 (7)

YSC1178-7501954 MATa DSL3-TAP::HIS3MX ura3Δ0 leu2Δ0 his3Δ met15Δ0 (7)

YSC1178-7500084 MATa SEC20-TAP::HIS3MX his3∆1 leu2∆0 met15∆0 ura3∆0 (7)

YSC1178-7500501 MATa TIP20-TAP::HIS3MX his3∆1 leu2∆0 met15∆0 ura3∆0 (7)

D3T-s18 MATa DSL3-TAP::HIS3MX sec18-1 ura3 his3 leu2 (8) D3T-d1 MATa DSL3-TAP::HIS3MX dsl1-22 ura3 his3 leu2 (8)

D3T-d1N MATalpha DSL3-TAP::HIS3MX dsl1Δ ura3 his3 leu2 + pRS315-DSLl1ΔN (8)

D3T-s20 MATalpha DSL3-TAP::HIS3MX sec20-1 ura3 his3 leu2 (8) D3T-t20/5 MATa DSL3-TAP::HIS3MX tip20-5 ura3 his3 leu2 (8) D3T-t20/8 MATa DSL3-TAP::HIS3MX tip20-8 ura3 his3 leu2 (8) CUY1416 MATa his3∆200 trp1∆63 ura3∆0 DSL3::TAP-KanMX6 this study CUY1500 MATa his3∆200 trp1∆63 ura3∆0 DSL1::TAP-KanMX4 this study

CUY1608 MATa his3∆200 trp1∆63 ura3∆0 ykt6∆::KanMX4 pRS424-YKT6pr-GFP-YKT6 this study

CUY1893 MATa his3∆200 trp1∆63 ura3∆0 DSL3::TAP-KanMX6 sec22∆::HIS3 this study

CUY2615 YSC1178-7501954 GAL1pr::pRS401-GAL1pr-YKT6 this study

CUY2710 MATa his3∆200 trp1∆63 ura3∆0 sec22pr::HIS3-GAL1pr DSL3::TAP-URA3 this study

CUY2848 MATa/alpha lys2∆0/LYS2 HIS3/his3∆1 LEU2/leu2∆0 MET15/met15∆0 URA3/ ura3∆0 TIP20/TIP20-TAP::HIS3MX this study

CUY2847 MATa/alpha lys2∆0/LYS2 HIS3/his3∆200 TRP1/trp1∆63 URA3/ura3∆0 DSL1/ DSL1-TAP::KanMX4 this study

CUY2925 BY4727 HIS3MX-PHO5pr-GFP::SEC20 this study CUY2976 BY4727 URA3::PHO5pr-GFP-SEC20 this study CUY2971 BY4741 sec22∆::KanMX4 HIS3MX:: PHO5pr-GFP-SEC20 this study

CUY2973 BY4741 KanMX4-GAL1pr::YKT6 pRS416-NOP1pr-YKT6 HIS3MX:: PHO5pr-GFP-SEC20 this study

CUY3007 RSY271 MATa sec18-1 his4-619 ura3-52 URA3::PHO5pr-GFP-SEC20 this study

CUY3010 MATa his3∆ leu2∆ ura3∆ dsl3-2 URA3::PHO5pr-GFP-SEC20 this study

CUY3027 BY4741 TIP20-TAP::HIS3MX DSL1-TAP::KanMX4 DSL3-TAP::URA3 this study

CUY2393 BY4741 TIP20:: monoGFP-KanMX4 this study CUY2394 BY4741 DSL3:: monoGFP-KanMX4 this study CUY1765 BY4741 DSL1:: monoGFP-KanMX4 this study

CUY4810 BY4727 DSL3::GFP-KanMX4 DSL1::GAL1pr hphNT1::SEC63-MARS this study

CUY4811 BY4727 DSL3::GFP-KanMX4 DSL1::GAL1pr hphNT1::MNN9-MARS this study

CUY4812 BY4727 SEC20::HIS3MX-PHO5pr-GFP DSL1::GAL1pr this study

hphNT1::SEC63-MARS

CUY4813 BY4727 SEC20::HIS3MX-PHO5pr-GFP DSL1::GAL1pr hphNT1::MNN9-MARS this study

XII-62 BY4727 DSL3::GFP-KanMX4 GAL-YPT1::LEU this study XIII-2 BY4727 SEC20::HIS3MX-PHO5pr-GFP GAL-YPT1::LEU this study

CUY5391 BY4727 SEC20::HIS3MX-PHO5pr-GFP ret2-1 hphNT1::SEC63-MARS this study

CUY6270 RSY271 MATa sec18-1 his4-619 ura3-52 URA3MX-PHO5pr-GFP-myc-tag::SEC20 SEC63::hphNT1-MARS this study

CUY6383 RSY271 MATa sec18-1 his4-619 ura3-52 SEC20::TAP-kanMX this study

CUY6384 RSY271 MATa sec18-1 his4-619 ura3-52 SEC20::TAP-kanMX YKT6::natNT2-GAL1pr this study

Table S2. Expression plasmids used for GST and His6-purification from E. coli.

Plasmid Backbone Insert Source/Reference pGEX-2TK DSL1 pGEX-2TK DSL1 (bp1-2265) this study pGEX-2TK DSL3 pGEX-2TK DSL3 (bp1-2130) this study pGEX-2TK TIP20 pGEX-2TK TIP20 (bp1-2106) this study pQLink H USE1-DSL3 pQLink USE1 (bp1-651) +

DSL3 (bp1-2130) (9)

pET32c(-Trx) UFE1 pET32c(-Trx) UFE1 (bp1-972) this study pET32c(-Trx) SEC20 pET32c(-Trx) SEC20 (bp1-825) this study pET32c(+) SEC20 NT pET32c(+) SEC20 (bp1-588) this study pET32c(+) SEC20 SD pET32c(+) SEC20 (bp571-825) this study pET32c(-Trx) SEC22 pET32c(-Trx) SEC22 (bp1-576) this study pETHIS-YKT6 pETHIS YKT6 (bp1-603) (10)

Table S3. Plasmids used in yeast two-hybrid analysis Plasmid Backbone Insert Source/Reference pACT2 – DSL1 pACT2 DSL1 (bp1-2265) this study pACT2 – DSL3 pACT2 DSL3 (bp1-2130) this study pACT2 – TIP20 pACT2 TIP20 (bp1-2106) this study pACT2 – TIP20 (1-221) pACT2 TIP20 (bp1-663) this study pACT2 – TIP20 (1-391) pACT2 TIP20 (bp1-1173) this study pACT2 – TIP20 (1-612) pACT2 TIP20 (bp1-1836) this study pACT2 – TIP20 (107-701) pACT2 TIP20 (bp319-2106) this study pACT2 – TIP20 (209-701) pACT2 TIP20 (bp624-2106) this study pACT2 – TIP20 (377-701) pACT2 TIP20 (bp1129-2106) this study pACT2 – TIP20 (611-701) pACT2 TIP20 (bp1831-2106) this study pACT2 – TIP20 (∆80-110) pACT2 TIP20 (Δbp238-330) this study pACT2 – UFE1 pACT2 UFE1 (bp1-972) this study pACT2 – UFE1 NT pACT2 UFE1 (bp1-719) this study pACT2 – UFE1 SD pACT2 UFE1 (bp709-972) this study pACT2 – USE1 pACT2 USE1 (bp1-654) this study pACT2 – USE1 NT pACT2 USE1 (bp1-423) this study pACT2 – USE1 SD pACT2 USE1 (bp418-654) this study pACT2 – SEC20 pACT2 SEC20 (bp1-588) this study pACT2 – SEC20 NT pACT2 SEC20 (bp1-588) this study pACT2 – SEC20 SD pACT2 SEC20 (bp571-825) this study pFBT9 – DSL1 pFBT9 DSL1 (bp1-2265) this study pFBT9 – DSL3 pFBT9 DSL3 (bp1-2130) this study pFBT9 – TIP20 pFBT9 TIP20 (bp1-2106) this study pFBT9 – SEC20 pFBT9 SEC20 (bp1-588) this study pFBT9 – USE1 pFBT9 USE1 (bp1-654) this study pFBT9 – SEC22 pFBT9 SEC22 (bp1-576) this study pFBT9 – YKT6 pFBT9 YKT6 (bp1-603) this study

SUPPLEMENTAL REFERENCES

1. Mayer, A., Wickner, W., and Haas, A. (1996) Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85, 83-94.

2. Dietrich, L.E., LaGrassa, T.J., Rohde, J., Cristodero, M., Meiringer, C.T., et al. (2005) ATP-independent Control of Vac8 Palmitoylation by a SNARE Subcomplex on Yeast Vacuoles. J Biol Chem 280, 15348-15355.

3. Tochio, H., Tsui, M.M., Banfield, D.K., and Zhang, M. (2001) An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science 293, 698-702.

4. Paravicini, G., Horazdovsky, B.F., and Emr, S.D. (1992) Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell 3, 415-427.

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10. Meiringer C.T., Auffarth K., Hou H., and Ungermann C. (2008) Depalmitoylation of Ykt6 prevents its entry into the multivesicular body pathway. Traffic 9(9),1510-1521.

50

40

30

2520

15

6070

85100120150200

Dsl3-cbp

Dsl1

Tip20

Ufe1

Sec20

Use1

Ykt6

50

40

30

2520

15

6070

85100120150200

8 9 10 11 12 13 14 15 16 17 18 19

A

37°C

B

Ufe1

Dsl1Dsl3-cbp

Tip20

Sec22

Sec22

Ykt6

Ykt6

Use1

D3T

D3T

D3T sec18

D3T se

c18

D3T D3T se

c18

23°C

23°C

23°C37°C

37°C

Input

Pulldown

Dsl3-TAPDsl1

Use1Ufe1

Tip20

Sec22

Ykt6

Dsl3-TAP

Dsl1

Use1

Ufe1Tip20

Sec22

Ykt6

1 2 3 4 5 6 7 8 9 10 11 12

+ATP

/Sec

18

C

DDsl3

Sec20

Use1

Ykt6

Dsl1

Dsl3

Tip20

Sec22

Tip20

Dsl1 Ufe1 Use1

Sec20

pFB

T9

n.d.

n.d.

Figure S1, Meiringer et al.

Ypt1

Vac8

0 9 9 0 9 9YPD YPG YPD YPG

GAL1-YPT1 wt

0 9 9 0 9 9YPD YPG YPDYPG

GAL1-YPT1 wt

hrs

Dsl3-GFP GFP-Sec20

E

~700kDa ~150kDaM W Dsl3-TA

P

fraction:

fraction:

pACT2

kDa kDa

Figure S2, Meiringer et al.

DIC MergeMARSDsl3-GFPA

B

Sec63-MARS

DIC MergeMARSGFP-Sec20

Mnn9-MARS

C0 06 9 96Sec63-MARS Mnn9-MARS

Dsl3-GFP

Dsl3

Dsl1

Arc1

hrs

YPD

YPG

YPD

YPG

GAL1-DSL1

Sec63-MARS

Mnn9-MARS

YPD

YPG

YPD

YPG

GAL1-DSL1

DIC Sec20-GFP MARS Merge

23°C

37°C

Sec63-MARS

D

Perz, Charles Barlowe, Hans Dieter Schmitt and Christian UngermannChristoph T.A. Meiringer, Ralf Rethmeier, Kathrin Auffarth, Joshua Wilson, Angela

snares - implications for fusion and fusion regulationThe Dsl1 tethering complex is a resident ER complex, which interacts with five

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