Sec1p and Sec4p suppress the growth and secretion defect of a sec3Δ mutant
Recent results have demonstrated that several tethering complexes can physically interact with the Rab proteins, SM proteins, and t-SNAREs that act in the corresponding fusion reaction (Guo et al., 1999
; Sato et al., 2000
; Seals et al., 2000
; Siniossoglou and Pelham, 2002
). For example, Sec4p was found to interact with the exocyst subunit Sec15p (Guo et al., 1999
). Furthermore, Sec4p, the exocyst, and Sec1p are all concentrated at sites of polarized secretion (Walch-Solimena et al., 1997
; Finger et al., 1998
; Carr et al., 1999
). We speculate that in addition to its function in membrane tethering, the exocyst may also bring together the various components of the exocytic machinery to facilitate exocytosis. Sec3p is the only nonessential subunit of the exocyst, and it plays a role mainly in targeting secretory vesicles to subdomains of the plasma membrane (Wiederkehr et al., 2003
). The absence of Sec3p also leads to a partial defect in exocytosis that may be the result of the inability of this mutant strain to concentrate essential protein components at sites of polarized secretion. Hence, overproduction of a limiting component might bypass the need for Sec3p or possibly other exocyst subunits in exocytosis. Therefore, we tested whether overproduction of Sec1p, Sec4p, or the t-SNAREs, Sso or Sec9p, could suppress the slow growth and partial secretion defect of a sec3
Multi-copy plasmids used to overexpress the genes of interest were introduced into a sec3Δ/SEC3 heterozygous diploid strain. The transformants were then sporulated and dissected. After dissection and marker analysis, wild-type and sec3Δ mutant haploids that retained the URA3 based multi-copy plasmid were struck out for single colonies on synthetic complete (SC)-Ura plates at 25°C. Overproduction of Sec1p or Sec4p clearly suppressed the growth defect of sec3Δ cells ( A and ). However, the suppressed sec3Δ strains remain temperature-sensitive at 37°C (unpublished data and ). As expected, a control strain overproducing Sec3p restored growth at all temperatures. An empty plasmid control ( A) or multi-copy plasmids carrying SEC5 or SEC6, encoding two other subunits of the exocyst, had no effect on sec3Δ growth. Interestingly, the multi-copy SSO2 or SEC9 plasmids also improved sec3Δ growth but less strikingly than either SEC1 or SEC4 ( A). These genetic results show that Sec1p, Sec4p and, to a lesser extent SNAREs, can compensate for the absence of Sec3p from the exocyst complex suggesting a functional connection between the exocyst, Sec1p, Sec4p, and SNARE proteins.
Figure 1. Sec1p and Sec4p overexpression stimulate growth and secretion of a sec3Δ mutant strain. (A) Wild-type (left) and sec3Δ mutant strains (right) carrying the indicated multi-copy (2μ) plasmids were grown on SC plates for 3 d at 25°C. (more ...)
Phenotypic characterization of the suppressed sec3Δ mutant strains
As mentioned earlier in this paper, sec3Δ cells have a partial defect in secretion. Therefore, we tested the extent to which the defect in the secretion of the derepressible, secreted protein invertase was suppressed when either Sec1p or Sec4p were overproduced. Overproduction of either Sec1p or Sec4p clearly improved secretion from a sec3Δ strain ( B). The sec3Δ strain accumulated 32% of the newly synthesized invertase in an intracellular pool, whereas in a wild-type strain only 13% was intracellular, corresponding to the amount of invertase in transit along the secretory pathway. In the sec3Δ strain overproducing Sec1p, the average value of intracellular invertase measured was 16%, which is not significantly different from the wild type. In the sec3Δ strain overproducing Sec4p, slightly more (18.5%) of the invertase was intracellular after derepression. Sec1p and Sec4p efficiently suppress the secretion defect of sec3Δ strains and allow them to grow at almost wild-type rates ( and ).
In an earlier study we found several phenotypes of sec3
Δ cells suggesting a defect in polarized secretion. Although Sec4p is concentrated in a very small area at the bud tip of wild-type cells, it is broadly distributed in the buds of sec3
Δ cells. Unlike the elongated wild-type cells, sec3
Δ cells are also round and are unable to extend normal mating projections (Wiederkehr et al., 2003
). Therefore, we tested whether overproduction of Sec1p or Sec4p, in addition to stimulating secretion, would also restore the polarity of sec3
Δ cells. The sec3
Δ cells overproducing Sec4p were round and showed defects in mating projection formation similar to sec3
Δ cells (, A–C). As Sec4p was overexpressed the Sec4p staining was stronger, but was still distributed broadly in the bud as in sec3
Δ cells (). In a wild-type background, Sec4p overexpression did not significantly affect the focal localization of Sec4p in the bud, although a fraction of the cells expressing very high levels of Sec4p showed additional cytoplasmic Sec4p staining. Surprisingly, overproduction of Sec1p led to a partial restoration of these sec3
Δ defects. A much larger fraction of sec3
Δ cells overproducing Sec1p were elongated, similar to the morphology of wild-type cells ( A). The sec3
Δ cells carrying the SEC1
multi-copy plasmid were also better at forming mating projections than sec3
Δ cells, although quite a few cells in the culture still showed aberrant, rounded projections (). Sec4p localization remained partially delocalized in sec3
Δ cells overexpressing Sec1p, but was more restricted at sites of polarized secretion than in the corresponding sec3
Δ strain (). It was surprising to find that overproduction of Sec1p restored secretion to a similar extent as Sec4p, yet unlike Sec4p also partially restored polarity. The final parameter we examined was the inheritance of cortical ER into the yeast bud. The sec3
Δ cells extend ER tubules into the bud, but the cortical ER fails to be established in the daughter cells. Overproduction of either Sec1p or Sec4p in the sec3
Δ cells failed to completely restore inheritance of the ER into the bud. Although most small buds still lacked cortical ER, in both cases overproduction did improve ER inheritance, as a significant fraction of the cells were able to establish cortical ER by the time the cells were large budded (). Tubule number, dynamics, and orientation appeared normal ().
Figure 2. Polarity defects of suppressed sec3Δ strains. (A) Percentage of yeast mother cells with wild-type-like elongated morphology was determined as described in Materials and methods. For each dataset the yeast strains were grown in parallel and DIC (more ...)
Figure 3. ER inheritance defects are partially restored in suppressed sec3Δ strains. (A) Hmg1-GFP (left) was used as a marker to follow the inheritance of the cortical ER during yeast bud growth of the indicated yeast strains. Corresponding DIC pictures (more ...)
Sec1p or Sec4p can bypass the requirement for Sec5p and Exo70p in exocytosis
Given the efficient suppression of the secretion defect of a sec3Δ mutant, we determined if overproduction of Sec1p or Sec4p could bypass the requirement for any other exocyst subunits. Dissection of sec6Δ/SEC6, sec8Δ/SEC8, sec10Δ/SEC10, sec15Δ/SEC15, or exo84Δ/EXO84 heterozygous diploids strains overproducing either Sec1p or Sec4p did not result in any viable haploid strains disrupted for these exocyst genes. However, dissection of sec5Δ/SEC5 and exo70Δ/EXO70 strains gave rise to viable haploid sec5Δ and exo70Δ strains in the presence of either the SEC1 or the SEC4 multi-copy plasmid ( A). The sec5Δ and exo70Δ strains were strictly dependent on Sec1p or Sec4p overproduction for viability. Tetrads in which the multi-copy plasmids were lost during sporulation only gave rise to two wild-type haploid strains. Furthermore, no sec5Δ and exo70Δ colonies were observed after selection against the URA3 plasmid marker on 5-fluoroorotic acid (5FOA) plates ( A). Only wild-type cells, which do not require the URA3-based plasmids, grew on SC plates containing 5FOA.
Figure 4. Sec1p or Sec4p overproduction can bypass a deletion of the essential exocyst genes SEC5 and EXO70. (A) Wild-type, sec5Δ, and exo70Δ strains were struck for single colonies on either SC or SC plates containing 5FOA (to select against the (more ...)
Sec1p and Sec4p are approximately equally efficient suppressors of the sec5Δ mutant ( A and ). However, in the case of the exo70Δ mutant, Sec1p was a clearly better suppressor than Sec4p ( A and ). The exo70Δ mutants overexpressing Sec1p grew very well, with a growth rate in liquid SC media close to that of the corresponding wild-type strain, and were not temperature sensitive for growth. In contrast, the exo70Δ mutant overexpressing Sec4p grows slowly both at 25 and 37°C. Both sec5Δ strains are tightly temperature sensitive for growth at 37°C ().
Phenotypic analysis of sec5Δ and exo70Δ mutant strains
We used invertase as a marker to measure the secretory defects of these strains. All of the suppressed mutants had only a weak secretory defect, accumulating 20–30% of the derepressed invertase in an intracellular pool ( B). Improvement of secretion by Sec1p or Sec4p is a likely explanation for the restoration of viability of the sec5Δ and exo70Δ mutants.
Although the exocyst works as a complex in secretion, specific subunits might confer different aspects of exocyst function. Therefore, we tested whether Sec5p and Exo70p, like Sec3p, are required for polarized secretion and ER inheritance. Cells lacking SEC5 have the broad Sec4p distribution and morphology defects observed for the sec3Δ cells (). The sec5Δ mutants also have a severe ER inheritance defect, similar to the sec3Δ strain. At each stage during bud growth, a large fraction of the sec5Δ cells have little or no cortical ER (), although the number of tubules is equal or higher than in the wild-type cells and tubule dynamics and orientation appear normal (). In summary, Sec5p appears to be as important for polarized secretion and ER inheritance as Sec3p. With regard to its Sec4p localization and morphology phenotypes, the exo70Δ strain overproducing Sec4p is similar to the sec3Δ and the suppressed sec5Δ mutant strains (). However, ER inheritance is only delayed in this mutant strain, as the defect is restricted to small budded cells (). By the time larger buds have formed, most exo70Δ cells have inherited cortical ER. Tubule number, dynamics, and orientation appear normal (). This distinction from the sec3Δ and sec5Δ mutants is even more striking in an exo70Δ mutant overproducing Sec1p, where ER inheritance is close to normal even in small budded cells (). In contrast, the sec3Δ and sec5Δ mutant overproducing Sec1p have very dramatic defects in ER inheritance, suggesting that the function of the Exo70p is less directly linked to ER inheritance than Sec3p and Sec5p. In addition, exo70Δ cells overproducing Sec1p are mostly elongated, similar to wild-type yeast cells ( A). Furthermore, the mating projections of the exo70Δ strain overproducing Sec1p are even more pronounced than those of the wild-type cells or wild-type cells overproducing Sec1p. Of all the mutants analyzed here, Sec4p localization was most highly polarized in the exo70Δ 2μSEC1 cells, although compared with wild-type cells, Sec4p was still partially delocalized (). Sec1p overproduction appears to improve polarized secretion, as in both the sec3Δ and exo70Δ mutant backgrounds Sec1p, but not Sec4p, clearly improves the morphology of the cells. The differences observed for the various strains, especially when overproducing Sec1p, show that Exo70p contributes differently to polarized secretion and ER inheritance than do Sec3p or Sec5p (). In summary, Sec5p and Exo70p carry out essential functions in the exocyst, but their function can be bypassed when secretion is stimulated by the overproduction of either Sec1p or Sec4p.
Figure 5. Analysis of polarity phenotypes of the suppressed sec5Δ and exo70Δ strains. The phenotypic analysis was performed as described in . (A) The percentage of elongated cells. (B) DIC images of mutant cells with shmoos. (C) Curvature (more ...)
Figure 6. Cortical ER inheritance in sec5Δ and exo70Δ mutant strains. The appearance of the ER marker Hmg1-GFP in the yeast bud during ER inheritance. (A) Hmg1-GFP fluorescence pictures (left) were taken at different stages of yeast bud growth. (more ...)
The sec3Δ, sec5Δ, and exo70Δ mutants show defects in exocyst assembly
Our working model of exocyst function is that the complex assembles to mediate vesicle tethering at the plasma membrane. By this model, the stably assembled exocyst assures that vesicles are tethered to the correct subdomain of the plasma membrane to allow the vesicles to undergo membrane fusion. Several of the temperature-sensitive exocyst mutants form a less stable complex or are missing specific subunits from the complex (TerBush and Novick, 1995
). We analyzed the assembly state of the exocyst in the deletion mutants. For this purpose endogenous Sec8p was myc epitope tagged and isolated from different mutant backgrounds. In the absence of Sec3p, Sec5p, or Exo70p there was a clear reduction in the yield of exocyst subunits that were coprecipitated with Sec8myc (). A large fraction of the exocyst complex (50–80%) was isolated by immunoprecipitation of Sec8myc from a wild-type strain ( A, lane 4). Although similar amounts of Sec8myc were isolated from a sec3
Δ strain, only 2–6% of Sec6p, Sec10p, or Sec15p was co-isolated ( A, lane 5). No background of exocyst subunits was observed when the isolation was conducted in parallel from an untagged control strain ( A, lane 3). These results demonstrate that in a sec3
Δ strain only a small fraction of the exocyst is assembled and sufficiently stable to be isolated by immunoprecipitation. Therefore, Sec3p is important for exocyst assembly or stability. Overproduction of Sec1p or Sec4p improves secretion in a sec3
Δ strain, but has no effect on the coprecipitation of the other exocyst subunits with Sec8myc ( A, lanes 6 and 7). Similar effects on exocyst assembly state were observed with the sec5
Δ and exo70
Δ mutants (). Only 2–6% of Sec6p, Sec10p, and Sec15p was co-isolated with Sec8myc from these strains, regardless of the suppressing plasmid ( C, lanes 6 and 12; D, lane 6). Overproduction of Sec1p or Sec4p in a wild-type background had no effect on exocyst isolation ( C, lanes 5 and 11).
Figure 7. Co-isolation of Sec1p and exocyst subunits with myc epitope tagged Sec8p. The exocyst was isolated from lysates of wild-type (B and controls in A, C, and D), sec3Δ (A), sec5Δ (C), and exo70Δ (D) mutant strains as described in Materials (more ...)
The above results concerning exocyst assembly in different mutant backgrounds are consistent with two possible interpretations. Either in these mutants Sec8myc binds more weakly to an otherwise fully assembled exocyst, or the absence of Sec3p, Sec5p, or Exo70p has a more global effect on the binding of exocyst subunits to each other. To distinguish between these possibilities, we also isolated the exocyst using a myc tag on Sec10p, another subunit of the exocyst. In a wild-type background, isolation of the exocyst using Sec10myc was similarly efficient as with Sec8myc. A large fraction of Sec6p, Sec8p, and Sec15p was co-isolated with the Sec10myc subunit from a wild-type lysate. However, ~10 times less Sec6p or Sec8p was co-isolated with Sec10myc from lysates of the different mutant strains (, lanes 5, 8, and 13). The results show that the exocyst is largely unassembled or less stably assembled in these mutant strains. Nonetheless, in all cases Sec10p still efficiently bound Sec15p. The amount of Sec15p in a Sec10myc immunoprecipitation was only slightly reduced in the sec3Δ, sec5Δ, and exo70Δ mutants relative to wild type (, lanes 5, 8, and 13). Therefore, Sec10p and Sec15p form a subcomplex that is little affected by the absence of Sec3p, Sec5p, or Exo70p from the complex. The abundance of Sec6p, Sec8p, Sec10p, and Sec15p in the lysate was not affected in the different mutants ( and ). Therefore, the absence of Sec3p, Sec5p, or Exo70p does not result in proteolysis of these other exocyst subunits. The results show that the assembly or stability of the complex is affected in these mutants, although a Sec10p–Sec15p subcomplex and possibly other subcomplexes remain intact.
Figure 8. Co-isolation of Sec1p and exocyst subunits with myc epitope–tagged Sec10p. The exocyst was isolated from a wild-type (lanes 6, 7, and 12), sec3Δ (lane 5), sec5Δ (lane 8), and exo70Δ (lane 13) mutant strain via a carboxy-terminal (more ...)
Sec1p binds to the exocyst
The Sec1p homologue Vps33p is a bona fide subunit of the class C/HOPS tethering complex required for vacuole-to-vacuole fusion (Sato et al., 2000
; Seals et al., 2000
). Given this result and the strong genetic interactions seen between SEC1
and exocyst mutants, we tested whether Sec1p is physically connected to the exocyst. We consistently observed that a minor fraction (0.2–0.4%) of Sec1p coprecipitated with the exocyst ( and ). The same amount of Sec1p was co-isolated with the exocyst from sec3
Δ cells and sec3
Δ, or exo70
Δ mutants suppressed by Sec4p overproduction, where only a small fraction of the exocyst is in its assembled state ( A, compare lane 4 with lanes 5 and 7; C, lanes 11 and 12; , lanes 7 and 8). Upon overproduction, an increased amount of Sec1p coprecipitated with the exocyst, although the relative fraction bound to the exocyst appeared to be very similar (, lanes 5 and 6; , lanes 5, 6, 12, and 13). When overproduced, the amounts of Sec1p bound to the exocyst were similar in sec3
Δ and wild-type lysates (, lanes 5 and 6). Compared with the wild type, even increased amounts of Sec1p were co-isolated in sec5
Δ and exo70
Δ mutant strains overproducing Sec1p (, lanes 5 and 6; , lanes 12 and 13). The coprecipitation was specific, as no signal above background was detected in myc precipitates from untagged strains expressing Sec1p at endogenous levels ( A, lane 3; C, lane 10) as well as from Sec1p-overproducing strains ( B, lane 4; , lane 14). Although binding of Sec1p to the exocyst is likely more transient than the interaction between Vps33p and the rest of the class C/HOPS complex, our results suggest a similar functional connection between these two tethering complexes and their corresponding SM family member.
Prior results from our laboratory showed that Sec1p binds to SNARE complexes (Carr et al., 1999
). Therefore, we tested whether the exocyst, possibly via its interaction with Sec1p, can associate with SNARE proteins. However, we could not detect any of the syntaxin-like SNARE Sso in the exocyst immunoprecipitates ( and ). These results imply that Sec1p can bind to the exocyst independent of assembled SNARE complexes.
Sec1p increases the levels of SNARE complexes
Members of the SM family bind to SNARE proteins and may regulate SNARE complex assembly, stability, or function (Kosodo et al., 2002
; Peng and Gallwitz, 2002
; Toonen and Verhage, 2003
; Scott et al., 2004
). Ongoing membrane traffic is essential for SNARE complex formation, and temperature-sensitive sec4
and exocyst mutants lead to the rapid loss of exocytic SNARE complexes after a shift to the restrictive temperature (Carr et al., 1999
; Grote et al., 2000
). Therefore, we tested how SNARE complex levels are affected by the absence of Sec3p, Sec5p, or Exo70p, as well as by Sec1p or Sec4p overproduction. For this analysis, steady-state levels of SNARE complexes were measured in the different mutants (). The SNARE complexes were isolated using an antibody against the v-SNARE Snc, and the relative amount of Sso in the immunoprecipitates was determined. Consistent with our earlier results, ~1% of Sso was co-isolated with Snc from a wild-type strain (Grote et al., 2000
). Upstream inhibition of membrane traffic and the concomitant slowed formation of SNARE complexes leads to a decrease in steady-state levels as Sec18p-mediated disassembly of SNARE complexes continues. As secretory function is only partially affected in the sec3
Δ mutant, SNARE complex levels were only reduced in this strain ( B, lane 6). In a sec3
Δ strain about half as much Sso was isolated in SNARE complexes together with Snc (40% ± 10; n
= 5), compared with a wild-type strain. Consistent with its ability to restore secretion, Sec4p overproduction also restored the amount of SNARE complexes that can be isolated from a sec3
Δ mutant background ( B, lane 8).
Figure 9. Sec1p overexpression increases SNARE complex levels. SNARE complexes were isolated from cleared yeast lysates by immunoprecipitation using the anti-Snc pAb as described in Materials and methods. The amount of Ssop bound to Sncp was compared by Western (more ...)
Surprisingly, overproduction of Sec1p not only restored the levels of SNARE complexes in a sec3Δ strain, but yielded complex levels that were actually about twofold higher (190% ± 30; n = 4) than in a wild-type strain ( B, lane 7). Such increased levels of SNARE complexes were also observed in sec5Δ and exo70Δ ( C, lane 5; D, lane 5). Importantly, increased SNARE complex levels were seen upon overproduction of Sec1p in a wild-type strain as well (201% ± 18, n = 5; A, lane 5), indicating that the effect is intrinsic to Sec1p overproduction rather than a response to the deletion mutations. Overproduction of Sec4p didn't have this effect in wild-type cells (118% ± 13, n = 3; A, lane 6). Also sec5Δ or exo70Δ strains overproducing Sec4p had SNARE complex levels similar to the wild-type strain (, lane 6).
In summary, the results with the sec3Δ strain show that overproduction of Sec4p can increase SNARE complex levels in a strain partially defective for tethering, presumably by restoring the flux of membrane through the pathway. However, Sec1p is more likely to play a direct role in SNARE function. The unexpected finding that SNARE complex levels are actually higher than normal in both mutant and wild-type strains overproducing Sec1p indicates that Sec1p can either increase assembly or slow disassembly of SNARE complexes. The bypass of the exocyst mutants sec3Δ, sec5Δ, and exo70Δ by Sec1p could be due to the increased SNARE complex levels under conditions where tethering is partially inhibited.
The Sec3, Sec5, and Exo70 proteins are apparently less essential for membrane traffic than the other five exocyst subunits. These results and the phenotypic analysis of the mutants described here show that different subunits are preferentially important for different aspects of exocyst function. As Sec1p binds to both the exocyst and SNARE complexes and can increase SNARE complex levels in vivo, we propose that Sec1p creates a functional link between exocyst-mediated vesicle tethering and SNARE complex–mediated vesicle docking and fusion.