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t-SNARE Activation Through Transient Interaction with a Rab-Like Guanosine Triphosphatase

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Science  23 May 1997:
Vol. 276, Issue 5316, pp. 1255-1258
DOI: 10.1126/science.276.5316.1255

Abstract

Intracellular vesicle targeting involves the interaction of vesicle proteins, termed v-SNAREs, with target membrane proteins, termed t-SNAREs. Assembly of v-SNARE–t-SNARE targeting complexes is modulated by members of the Sec1-Sly1 protein family, and by small guanosine triphosphatases termed Rabs. The interactions of these proteins during assembly of the endoplasmic reticulum–to-Golgi targeting complex inSaccharomyces cerevisiae were studied. The data suggest that the Rab protein Ypt1p transiently interacts with the t-SNARE Sed5p and results in displacement of the negative regulator Sly1p, allowing subsequent formation of the v-SNARE–t-SNARE targeting complex.

The movement of proteins between intracellular membrane-bounded compartments is mediated by transport vesicles that bud from one compartment and target specifically to the next compartment (1). Vesicle targeting involves interactions between integral membrane proteins, the v-SNAREs and t-SNAREs, that reside on vesicle and target membranes, respectively (2, 3). The v/t-SNARE complex then binds soluble NSF attachment protein [SNAP, Sec17p in yeast (4)] andN-ethylmaleimide–sensitive fusion protein [NSF, Sec18p in yeast (5, 6)], which catalyze the disassembly of the v/t-SNARE complex as a prelude to membrane fusion (7). Rab proteins, a family of small guanosine triphosphatases (GTPases) related to Ras, also act in vesicle targeting but their precise role is unclear (8).

Targeting of vesicles from the endoplasmic reticulum (ER) to the Golgi in yeast involves three v-SNAREs (Sec22p, Bet1p, and Bos1p) (9,10), the t-SNARE Sed5p (11), the Rab-like GTPase Ypt1p (12, 13), and a peripheral membrane protein termed Sly1p (14) that associates with Sed5p (10). Although Sly1p is required for ER to Golgi transport (15, 16), it may also act as a negative regulator (17).

To examine whether Ypt1p physically interacts with Sed5p, we immunoblotted Sed5p immunoprecipitates (IPs) from strains (18) with and without a high copy number (2 μm)YPT1 plasmid (19) (Fig. 1A). A weak Ypt1p-Sed5p interaction was detected in wild-type cells, which was greatly enhanced by overexpression of Ypt1p. The Ypt1p-Sed5p interaction appeared to be specific because immunoprecipitation of the v-SNARE Sec22p from the same lysates did not yield detectable Ypt1p. All of the Sed5p or Sec22p was immunoprecipitated from the lysates, yet only a small fraction of the Ypt1p was recovered with the Sed5p. This suggests that the Ypt1p-Sed5p interaction is of low affinity or is transient.

Figure 1

Physical interaction of Ypt1p with Sed5p. (A) In vivo protein interaction. Spheroplasts of RSY255 (lanes 1, 3, 5, and 7) or RSY255 pNB167 (YPT1,URA3, 2 μm) (lanes 2, 4, 6, and 8) were lysed with nonionic detergent and proteins immunoprecipitated (IP) with antibody to Sed5p (anti-Sed5p) or anti-Sed22p (28). The IPs (lanes 1 to 4) and one-fifth of the corresponding supernatants (lanes 5 to 8) were then immunoblotted to detect Sed5p, Sec22p, and Ypt1p. (B) In vitro interaction. Ten micrograms of GST-Sar1p (∼200 pmol), GST-Sec22p (∼200 pmol), or GST-Sed5p (∼150 pmol) in phosphate-buffered saline (PBS), or PBS alone (blank), was mixed with 0.2 μg of His6-Ypt1p (∼10 pmol) in 1 ml of buffer B [20 mM Hepes-KOH (pH 7.0), 150 mM potassium acetate, 0.5 mM dithiothreitol, 0.05% Tween-20], incubated for 2 hours at 4°C, and centrifuged at 15,000g for 15 min. The supernatant (0.95 ml) was incubated for 1 hour at 4°C with 40 μl of glutathione–Sepharose 4B equilibrated in buffer B. Beads were washed four times at 23°C with 1 ml of buffer B and transferred to another tube. Bound proteins were eluted with 20 μl of 10 mM reduced glutathione in 50 mM tris-HCl (pH 7.5), separated by SDS-PAGE, transferred onto nitrocellulose, stained with Ponceau S [which indicated that equivalent amounts of GST-fusion protein were recovered (23)], and immunoblotted with antibodies (28) to Ypt1p. About 8% of the His6-Ypt1p present in the reaction was recovered with the GST-Sed5p beads.

To determine if the in vivo interaction of Sed5p with overexpressed Ypt1p was direct, we attempted to reconstitute this interaction with purified proteins (Fig. 1B). His6-tagged Ypt1p (His6-Ypt1p) was incubated with buffer, or with glutathione-S-transferase (GST)–Sar1p, GST-Sec22p, or GST-Sed5 fusion proteins, and the complexes were recovered on glutathione-Sepharose beads. Sar1p participates in the formation of ER-derived coat protein II (COP II)–coated vesicles (20), thus GST-Sar1p serves as a negative control. His6-Ypt1p did not bind to GST-Sar1p, whereas a low but reproducible amount of His6-Ypt1p was bound to GST-Sec22p. In contrast, His6-Ypt1p bound efficiently to GST-Sed5p. This result corroborates the Sed5p-Ypt1p interaction observed in vivo and shows that the Sed5p-Ypt1p interaction is direct.

To explore whether the physical association of overexpressed Ypt1p with Sed5p influences the assembly of v/t-SNARE complexes in vivo, we immunoblotted Sed5p IPs from lysates of various mutant strains to determine if other components of the v/t-SNARE complex were present (Fig. 2A). As previously shown (10, 21), the v-SNAREs Sec22p and Bet1p were not associated with Sed5p in wild-type cells, probably because of the transient nature of the v/t-SNARE complex. In contrast, Sec22p and Bet1p were associated with Sed5p in asec18 strain, which lacks NSF activity at the restrictive temperature (6) and therefore cannot disassemble the v/t-SNARE complex (7, 10, 21). The ypt1 strain (22) at the restrictive temperature accumulated no v/t-SNARE complex. This result has been previously interpreted to suggest that Ypt1p is required for v/t-SNARE complex assembly (10). For a more rigorous test, we constructed a ypt1 sec18double-mutant strain. If Ypt1p is required for v/t-SNARE complex assembly, then the ypt1 defect should block thesec18-dependent v/t-SNARE complex accumulation. We found this to be the case: Less Sec22p and Bet1p were associated with Sed5p in the ypt1 sec18 double-mutant strain than in thesec18 strain. Assembly of v/t-SNARE complexes in theypt1 sec18 strain was restored by YPT1 on a low copy number centromere-based (CEN) plasmid, confirming that reduced assembly in the double mutant was due to the ypt1 mutation. Complementation with the high copy number 2-μm YPT1plasmid was not as effective as that with the CEN plasmid and correlated with the association of Ypt1p with Sed5p. This Ypt1p-Sed5p interaction was apparently specific because it was dependent on immunoprecipitation with the Sed5p antibody; beads without antibody bound neither Sed5p nor Ypt1p from the same strain. These data confirm that Ypt1p is required for v/t-SNARE complex assembly in vivo (10) and suggest that large amounts of Ypt1p can interfere with the assembly of v/t-SNARE complexes by binding to Sed5p.

Figure 2

Inhibition of v- and t-SNARE interaction after overexpression of Ypt1p. (A) Sed5p, Sec22p, Bet1p, and Ypt1p immunoblots of anti-Sed5p IPs from nonionic detergent–lysed spheroplasts shifted to 38°C for 20 min before lysis. (B) Same as (A) for anti-Sec22p IPs. Lane 1, RSY255 (WT); lane 2, RSY271 (sec18-1); lane 3, RSY976 (ypt1-3); lane 4 GWY141 (sec18-1 ypt1-3); lane 5, GWY141 pNB166 (YPT1, URA3, CEN); lane 6, GWY141 pNB167 (YPT1, URA3, 2 μm); lane 7, as in lane 6, but mock-immunoprecipitated without antibodies to Sed5p. The polypeptide evident above Sed5p is the heavy chain of the immunoprecipitating antibody (Ab HC). The method was as described (29) except that the spheroplasts were regenerated at 38°C. (C) Sed5p and Sec22p immunoblots of Sed5p IPs (29). Lane 1, RSY271 (sec18-1); lane 2, GWY141 (sec18-1 ypt1-3); lane 3, GWY141 pYCP50-SLY1-20 (SLY1-20, URA3, CEN); lane 4, GWY141 pSLY1-20 (SLY1-20,URA3, 2 μm).

We also analyzed assembly of the v/t-SNARE complex by immunoprecipitation of Sec22p (Fig. 2B). The results indicated again that Ypt1p is required for v/t-SNARE complex assembly and that overexpression of Ypt1p inhibits the process. A small amount of overexpressed Ypt1p immunoprecipitated with Sec22p (Fig. 2B), but the amount was much less than that found in Sed5p IPs (Fig. 2A).

In all cases, Bet1p only associated with Sec22p under conditions of v/t-SNARE complex assembly (Fig. 2B), suggesting that the v-SNARE Bet1p only stably associates with the v-SNARE Sec22p in the v/t-SNARE complex. Association of the v-SNARE Bos1p with Sec22p was also enhanced in a sec18 strain (10, 23), suggesting that these v-SNAREs interact tightly with one another only in the v/t-SNARE complex.

The absence of Ypt1p activity in vivo leads to a growth defect and a block in ER to Golgi transport (12). These phenotypes can be suppressed by the presence of a dominant mutant allele ofSLY1, termed SLY1-20 (14, 15).SLY1-20 can also bypass the requirement for Ypt1p in v/t-SNARE complex assembly, because expression of Sly1-20p from a low copy number (CEN) plasmid in the ypt1 sec18 double-mutant strain (Fig. 2C) almost completely restored v/t-SNARE complex assembly to the level obtained in the sec18 strain; larger amounts of Sly1-20p expressed from a 2-μm plasmid completely restored v/t-SNARE complex assembly. The correlation of the SLY1-20 genetics (14, 15) with these results suggests that the immunoprecipitation technique faithfully measures v/t-SNARE complex assembly in vivo.

It has been proposed that Sly1p is a component of the yeast ER-Golgi v/t-SNARE complex (10). In contrast, the Sly1p homolog involved in synaptic vesicle docking, n-Sec1 (24), is not a v/t-SNARE complex component (2). We probed the association of a v-SNARE, the t-SNARE, and Sly1p with one another by immunoprecipitation under conditions that favor v/t-SNARE complex assembly, that is, in a sec18 strain at the restrictive temperature (Fig. 3A). Both Sly1p and Sec22p were present in the Sed5p immunoprecipitate, but Sec22p was excluded from the Sly1p immunoprecipitate, and little Sly1p was in the Sec22p immunoprecipitate. This finding suggests that Sed5p is present in at least two different complexes: a Sed5p-Sly1p complex and a Sed5p-Sec22p complex, and that the v/t-SNARE complex does not contain Sly1p.

Figure 3

Absence of Sly1p from v/t-SNARE complexes. (A) Sec22p, Sed5p, and Sly1p immunoblots of Sec22p, Sed5p, and Sly1p IPs (lanes 1 through 3) and one-fifth of the supernatants (lanes 4 to 6) from nonionic detergent-lysed RSY271 (sec18-1) spheroplasts. The method was as described (29) except that the spheroplasts were regenerated for 20 min at 38°C. (B) Sed5p and Sly1p immunoblots of denaturing Sed5p IPs from cross-linked lysates made from RSY255 (WT) and RSY271 (sec18-1) spheroplasts incubated at the permissive (23°C) and restrictive (38°C) temperature (T). Methods are described in (30).

We also studied the Sed5p-Sly1p complex by cross-linking these proteins in yeast lysates (Fig. 3B). In lysates from wild-type cells, comparable amounts of Sly1p were cross-linked to Sed5p at 23° and 38°C. In asec18 strain, under conditions of v/t-SNARE complex accumulation (38°C), less Sly1p was cross-linked to Sed5p. Apparently, a large fraction of Sed5p was no longer associated with Sly1p, presumably because the Sed5p was present in v/t-SNARE complexes at the sec18 restrictive temperature. Again, these results suggest that Sly1p is not a stable component of the yeast ER-Golgi v/t-SNARE complex, and that the interaction of Sly1p with Sed5p can be modulated.

The findings that Sly1p interacts with Sed5p before, but not after, formation of v/t-SNARE complexes, and that Ypt1p transiently interacts with Sed5p before assembly, prompted us to test whether Ypt1p influences the Sed5p-Sly1p interaction (Fig. 4). Different amounts of Ypt1p were expressed in a sec18 strain with a regulatable GAL10 promoter (sec18PGal-YPT), and proteins immunoprecipitating with Sed5p were examined by immunoblotting. A sec18 strain was included as a control. Repression of Ypt1p synthesis in glucose-rich media precluded SNARE complex assembly, as indicated by the reduced association of the v-SNARE Bet1p with the t-SNARE Sed5p. Synthesis of approximately wild-type amounts of Ypt1p in raffinose media allowed SNARE complex assembly, whereas production of large amounts of Ypt1p in galactose-containing media less effectively promoted SNARE complex assembly. Examination of the amount of Sly1p associated with Sed5p at various Ypt1p levels showed that the Sed5p-Sly1p interaction was inversely proportional to the cellular amount of Ypt1p. This suggests that Ypt1p acts to disrupt or lower the affinity of the Sed5p-Sly1p interaction.

Figure 4

Sensitivity of the Sed5p-Sly1p complex to the amount of Ypt1p. (Three upper panels) Sed5p, Bet1p, and Sly1p immunoblots of Sed5p IPs (29) fromsec18 cells incubated at the restrictive temperature and expressing various amounts of Ypt1p. (Bottom panel) Ypt1p immunoblot of the corresponding lysates. GWY142 (sec18-1PGal-YPT1) was grown to mid-logarithmic phase in YP media (29) containing 2% raffinose, pelleted, resuspended in fresh YP media containing either 4% glucose (Glu, lane 1), 2% raffinose (Raf, lane 2), or 2% galactose (Gal, lane 3), and grown for 4 hours at 23°C. RSY271 (sec18-1) was grown on YP and 4% glucose (Glu, lane 4). Spheroplasts were prepared and processed as described (29) except that they were regenerated for 60 min at 38°C in the same media in which they were grown supplemented with 0.8 M sorbitol. Controls without PGal-YPT1indicated that the various levels of Sed5p-Sly1p interaction were not a function of carbon source (23).

These results lead us to suggest a model for assembly of the v/t-SNARE vesicle-targeting complex that includes a specific role for the Rab-like GTPase Ypt1p (Fig. 5). We propose that before association of Sed5p with Sly1p, the t-SNARE is not competent for v-SNARE interaction (25). Association of Sly1p with Sed5p is required for subsequent t-SNARE functionality (26), but at the same time, Sly1p keeps the t-SNARE in an inactive state (17); this would explain both the positive and negative effects on transport of the Sly1p family. The displacement of Sly1p by Ypt1p, which may arrive at the docking site in association with the transport vesicle (13, 27), renders the t-SNARE competent for v-SNARE interaction. The interaction of Ypt1p with Sed5p is transient, and after, or perhaps concomitant with, its dissociation the activated Sed5p engages the v-SNAREs, resulting in an assembled v/t-SNARE complex. Sec17p and Sec18p (SNAP and NSF) can then interact with the assembled complex and catalyze disassembly (7), allowing membrane fusion to occur.

Figure 5

Model for the regulation of v/t-SNARE complex assembly.

  • * To whom correspondence should be addressed. E-mail: gwaters{at}molbiol.princeton.edu

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