Synthetic Interactions of the Post-Golgi sec Mutations of Saccharomyces cerevisiae

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1 Copyright 2000 by the Genetics Society of America Synthetic Interactions of the Post-Golgi sec Mutations of Saccharomyces cerevisiae Fern P. Finger 1 and Peter Novick Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut Manuscript received March 21, 2000 Accepted for publication July 3, 2000 ABSTRACT In the budding yeast Saccharomyces cerevisiae, synthetic lethality has been extensively used both to characterize interactions between genes previously identified as likely to be involved in similar processes as well as to uncover new interactions. We have performed a large study of the synthetic lethal interactions of the post-golgi sec mutations. Included in this study are the interactions of the post-golgi sec mutations with each other, with mutations affecting earlier stages of the secretory pathway, with selected mutations affecting the actin cytoskeleton, and with selected cell division cycle (cdc) mutations affecting processes thought to be important for or involving secretion, such as polarity establishment and cytokinesis. Synthetic negative interactions of the post-golgi sec mutations appear (as predicted) to be largely stage specific, although there are some notable exceptions. The significance of these results is discussed in the context of both secretory pathway function and the utility of synthetic lethality studies and their interpretation. assessing whether gene products are involved in similar cellular processes, and it has been exploited in screens for new mutations affecting similar biological processes (Bender and Pringle 1991). In the secretory pathway in yeast (Kaiser et al. 1997), synthetic lethality has been used extensively both to characterize interactions between genes previously identified as likely to be involved in similar processes as well as to uncover new interac- tions. This pathway begins with the cotranslational trans- location into the endoplasmic reticulum (ER) of pro- teins destined for export. Proteins begin to be modified in the ER and are subsequently transported to the Golgi complex via small vesicle carriers. In the Golgi, proteins are further modified, and then sorted from proteins that will reside in the vacuole, and transported via post- Golgi secretory vesicles that fuse with the plasma mem- brane. A combination of phenotypic and genetic analy- ses has divided this pathway into several stages: the formation of vesicles from the ER, the fusion of these vesicles with the Golgi, intra-golgi transport, and the fusion of post-golgi vesicles with the plasma membrane. We present here a study of the synthetic lethal interactions and synthetic negative genetic interactions of one class of yeast secretory mutations, the post-golgi sec mu- tations. These are temperature-sensitive alleles of the SEC1, SEC2, SEC3, SEC4, SEC5, SEC6, SEC8, SEC9, SEC10, and SEC15 genes (Novick et al. 1981). Their gene prod- ucts include several members of a large complex re- quired for vesicle tethering at the plasma membrane, a rab family GTPase and its accessory proteins, and SNAREs and their regulators. This study comprises the interactions of the post-golgi sec mutations with each other, with mutations affecting earlier stages of the secretory pathway, with selected mutations affecting the actin cytoskeleton, and with selected cell division cycle (cdc) mutations affecting processes thought to be impor- THE term synthetic lethality was first used by Dobzhansky (1946) to describe the phenomenon where alleles of different genes are separately viable, but inviable when combined in a double mutant. Such interactions can be interpreted in several ways, depending upon the characteristics of the interacting al- leles (reviewed in Guarente 1993). For example, if both mutations are null, the interpretation is that the genes are required in parallel pathways with a common essential function, loss of which is lethal. This type of interaction is frequently the result of genetic redun- dancy. In cases where single null mutations are lethal, mutations with partial function must be used to evaluate synthetic phenotypes. Synthetic lethality in such cases is then usually interpreted as indicating that both genes function in the same essential pathway. Such synergy could result from successive reductions in flow through the pathway at different discrete stages such that the lower throughput is the product of reduced efficiency of the two steps. More frequently, synthetic lethality is interpreted as resulting from genes affecting the same stage of a pathway, as when mutations weaken the inter- actions of subunits of a complex, although a synthetic lethal interaction does not necessarily imply a physical interaction. In the budding yeast Saccharomyces cerevisiae, synthetic lethality was first noted in studies of the allele specificity of suppressors of actin mutations (Novick et al. 1989). It has subsequently been used extensively as a means of Corresponding author: Peter Novick, Department of Cell Biology, Yale University School of Medicine, P.O. Box , New Haven, CT peter.novick@yale.edu 1 Present address: Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Dr., Madison, WI Genetics 156: (November 2000)

2 944 F. P. Finger and P. Novick tant for or involving secretion, such as polarity establish- severe alleles sec1-13, sec3-4, and sec3-5 are all synthetically ment and cytokinesis. A number of these interactions lethal with sec6-4 (Figure 1). have been analyzed in earlier publications (Salminen SNAREs and their regulators form the second subcategory and Novick 1987; Nair et al. 1990; Bowser et al. 1992; of post-golgi sec mutations. We have also included Potenza et al. 1992; Moya et al. 1993; Govindan et al. here sec17-1 and sec18-1, which are mutations defective 1995); here we extend, and in some cases reexamine, for the yeast NSF and -SNAP homologs, respectively this analysis. (Wilson et al. 1989; Griff et al. 1992). Although the observed secretory block for these two mutations is in ER-to-Golgi transport (Novick et al. 1981), we include MATERIALS AND METHODS them here as they are also required for post-golgi secretion and physically interact with yeast post-golgi The S. cerevisiae strains used in this study are listed in Table 1. For each post-golgi sec mutation, at least one tempera- SNAREs (Brennwald et al. 1994; Graham and Emr ture-sensitive (ts) allele was used (sec2-41, sec4-8, sec5-24, sec6-4, 1991). The other members of this category are the two sec8-9, sec9-4, sec10-2, and sec15-1). In the cases of sec1, two alleles of the gene encoding the SNARE interacting alleles were used (sec1-1 and sec1-13), and for sec3, three alleles protein Sec1p (Carr et al. 1999), sec1-1 and sec1-13, were used (sec3-2, sec3-4, and sec3-5). Standard procedures were and an allele of the SNAP-25 homolog Sec9p, sec9-4 used for yeast growth, mating, and genetics (Guthrie and Fink 1991). Matings were usually performed using complemutations display negative synthetic interactions with (Brennwald et al. 1994). We find that sec1 and sec9 mentation of auxotrophic markers to select for diploids. Where auxotrophic selection was not available, complementa- each other and with the other post-golgi sec mutations tion of ts growth was used. Yeast were generally grown prior (Figure 2). No interactions were observed in crosses to sporulation on YPD plates; however, strains that did not with sec17-1 and sec18-1 (Figure 2). sporulate efficiently on YPD were grown on presporulation plates. All strains were sporulated at 25 on sporulation methose affecting the rab-family GTPase, Sec4p (Salminen The last grouping of post-golgi sec mutations are dium containing 1% potassium acetate, 0.5% glucose, 0.2% raffinose, and 0.1% yeast extract. Growth phenotypes were and Novick 1987), and its accessory proteins. These assessed following replica plating of colonies that germinated include the guanine nucleotide exchange factor (GEF), at 25 onto YPD plates at 25, 30, 34, and 37. Where alleles Sec2p (Walch-Solimena et al. 1997), the GDP dissociawere tagged with auxotrophic markers, testing for the prestion inhibitor Gdi1p (allelic with SEC19; Garrett et al. ence of the marker was also by replica plating. In most cases, the presence of double mutants was inferred from the growth 1994), and the guanine-nucleotide releasing protein, phenotypes of the other colonies in the tetrads. A minimum Dss4p (Moya et al. 1993). We find that sec4-8 and of 9 informative tetrads (those containing double mutants) sec2-41 interact with all of the other post-golgi sec mutaout of at least 12 tetrads dissected were obtained for each tions (Figure 3). In contrast, the interactions of sec19-1 cross. This was considered to be sufficient for cases in which no interaction was observed. In most cases at least 12 or more and dss4 are more specific. There is no negative syn- informative tetrads were obtained, particularly in instances thetic interaction between either sec9-4 or sec15-1 and where results were contrary to expectations. Interactions were sec19-1. dss4 interacts only with sec2-41, sec3-2, sec3-5, considered to be significant if double mutants were dead or sec4-8, sec9-4, and sec15-1, but not with sec1-1, sec1-13, their growth dramatically reduced at 25, or if the restrictive sec3-4, sec5-24, sec6-4, and sec10-2 (Figure 3). temperature was reduced by at least 3. Interaction of post-golgi sec mutations with those affecting earlier stages of the secretory pathway: We next RESULTS crossed the post-golgi sec mutants to those affecting ERto-Golgi and intra-golgi transport (Figure 4). With one Interaction of the post-golgi secretory mutations with striking exception we see no strong interaction of any each other: We first looked at the interactions of the of the post-golgi sec mutations with the sec mutations post-golgi sec mutations with each other, dividing them affecting either ER-to-Golgi transport (sec12-4, sec13-1, into three functional categories. Several of the post- sec16-2, sec20-1, sec21-1, sec22-3, and sec23-1), or with Golgi sec gene products, those encoded by SEC3, SEC5, those affecting intra-golgi transport (sec7-1 and sec14- SEC6, SEC8, SEC10, and SEC15, are components of a 3). This notable exception is the lowering of the temperlarge complex that is peripherally associated with and ature at which lethality occurs from 37 to 30, compared involved in vesicle tethering at the plasma membrane to both single mutants, in the sec14-3 sec15-1 double (Bowser et al. 1992; TerBush and Novick 1995; Ter- mutant. SEC14 encodes a phosphatidylinositol/phos- Bush et al. 1996; Finger et al. 1998; Guo et al. 1999). phatidylcholine transfer protein (Bankaitis et al. 1990). When the post-golgi sec mutations are crossed with The YPT1 gene product has been implicated in transmutations in the components of this tethering com- port from the ER to the Golgi and in intra-golgi transplex, we find that most combinations are synthetically port (Bacon et al. 1989; Jedd et al. 1995). We crossed lethal or lower the restrictive temperature, regardless four mutant alleles of YPT1 to the post-golgi sec mutants. of whether both components are complex members Three of these alleles are ts (ypt1-1, ypt1-3, and ypt1 I121,V161 ; (Figure 1). The sole exceptions to this are that neither Segev and Botstein 1987; Schmitt et al. 1988; Wuestehube sec1-1 nor sec3-2 interacts with sec6-4, although the more et al. 1996), and one of them (ypt1-1) is quite

3 Synthetic Lethality of Late sec Mutants 945 TABLE 1 Strains used TABLE 1 (Continued) Strain Genotype Strain Genotype NY3 MATa ura3-52 sec1-1 NY431 MATa ura3-52 sec18-1 NY4 MAT his4-619 sec1-1 NY432 MAT ura3-52 sec18-1 NY5 MATa his4-619 sec1-1 NY435 MATa ura3-52 ypt1-1 NY8 MAT ura3-52 sec1-1 NY703 MAT ura3-52 ypt1-2 NY16 MAT his4-619 sec6-4 NY705 MATa his4-619 ypt1-2 NY17 MATa ura3-52 sec6-4 NY706 MAT his4-619 ypt1-2 NY21 MAT ura3-52 sec6-4 NY707 MATa ura3-52 ypt1-2 NY54 MATa his4-619 sec9-4 NY737 MAT ura3-52 leu2-3,112 sec23-1 NY55 MAT his4-619 sec9-4 NY738 MATa ura3-52 sec12-4 NY57 MATa ura3-52 sec9-4 NY757 MATa his4-619 sec7-1 NY58 MAT ura3-52 sec9-4 NY760 MAT ura3-52 sec7-1 NY60 MATa ura3-52 his4-619 sec10-2 NY761 MAT his4-619 sec7-1 NY61 MATa ura3-52 sec10-2 NY769 MATa leu2-3,112 sec1-1 NY62 MAT ura3-52 his4-619 sec10-2 NY771 MATa leu2-3,112 sec2-41 NY63 MAT his4-619 sec10-2 NY777 MATa leu2-3,112 sec5-24 NY64 MATa ura3-52 sec15-1 NY780 MAT ura3-52 leu2-3,112 sec8-9 NY65 MAT his4-619 sec15-1 NY782 MATa ura3-52 leu2-3,112 sec9-4 NY66 MAT ura3-52 sec15-1 NY785 MATa leu2-3,112 sec10-2 NY67 MATa his4-619 sec15-1 NY786 MATa ura3-52 leu2-3,112 sec15-1 NY130 MATa ura3-52 sec2-41 NY869 MATa leu2-3,112 ypt1-1 NY132 MAT ura3-52 his4-619 sec2-41 NY1006 MATa ura3-52 leu2-3,112 myo2-66 NY133 MAT his4-619 sec2-41 NY1214 MAT leu2-3,112 sec19-1 NY181 MATa sec14-3 NY1215 MATa leu2-3,112 sec19-1 NY276 MAT his4-619 act1-1 NY1221 MAT ura3-52 sec3-2 NY278 MAT ura3-52 act1-1 NY1222 MATa ura3-52 sec3-2 NY399 MAT ura3-52 sec5-24 NY1224 MAT ura3-52 leu2-3,112 sec3-2 NY400 MATa his4-619 sec5-24 NY1225 MAT leu2-3,112 sec3-2 NY401 MAT his4-619 sec5-24 NY1226 MATa leu2-3,112 sec3-2 NY402 MATa ura3-52 sec5-24 NY1258 MATa lys2-801 sec3-2 NY404 MAT his4-619 sec4-8 NY1260 MATa leu2-3,112 ypt1-3 NY405 MATa ura3-52 sec4-8 NY1263 MAT ura3-52 ypt1-3 NY407 MAT ura3-52 sec4-8 NY1265 MAT leu2-3,112 ypt1-3 NY409 MATa his4-619 sec4-8 NY1294 MATa leu2-3,112 sec6-4 NY410 MATa ura3-52 sec8-9 NY1339 MAT leu2 ura3-52 ypt1 I121,V161 -LEU2 NY411 MAT his4-619 sec8-9 NY1449 MAT ura3-1 leu2-3,112 his3-11,15 ade1 NY412 MATa ura3-52 sec3-2 ade2 ade3-22 trp1-1can1-100 cdc28-1 NY413 MAT ura3-52 sec13-1 NY1636 MATa lys2-801 his3 cdc24-4 NY414 MATa ura3-52 sec13-1 NY1640 MATa lys2-801leu2 cdc42-1 NY415 MATa ura3-52 sec16-2 NY1644 MAT his4-619 cdc28-1 NY416 MAT ura3-52 sec16-2 NY1649 MATa leu2-3,112 cdc12-6 NY417 MAT ura3-52 sec17-1 NY1654 MATa leu2-3,112 cdc28-1 NY418 MATa ura3-52 sec17-1 NY1655 MAT lys2-801 cdc42-1 NY419 MAT ura3-52 sec19-1 NY2014 MATa ura3-52 sec1-13 NY420 MATa ura3-52 sec19-1 NY2015 MATa his4-619 sec1-13 NY421 MAT ura3-52 sec20-1 NY2018 MAT ura3-52 sec1-13 NY422 MATa ura3-52 sec20-1 NY2020 MAT ura3-52 sec1-13 NY423 MATa ura3-52 leu2-3,112 sec21-1 NY2098 MAT ura3-52 sec3-4 NY424 MAT ura3-52 sec21-1 NY2099 MATa ura3-52 sec3-4 NY425 MATa ura3-52 sec20-1 NY2108 MAT leu2-3,112 sec3-4 NY426 MATa ura3-52 sec22-3 NY2115 MATa ura3-52 sec3-5 NY427 MAT ura3-52 leu2-3,112 trp1 his4 sec12-4 NY2118 MAT ura3-52 sec3-5 NY428 MAT ura3-52 leu2-3,112 his3 sec23-1 SFNY330 MAT leu2 his3 ypt1, I121,V161 -LEU2 NY429 MAT ura3-52 sec14-3 SFNY446 MATa ura3-52 ypt1-3 NY430 MATa ura3-52 sec14-3 SFNY447 MAT ura3-52 leu2-3,112 ypt1-3 (continued)

4 946 F. P. Finger and P. Novick Figure 1. Crosses of post-golgi sec mutants to mutants in members of the tethering complex. White boxes indicate no interaction, black boxes indicate synthetic lethality at 25, and gray boxes indicate that the restrictive temperature of the double mutant was at least 3 lower than that of either single mutant. AL, the mutations are allelic; ND, not determined. Data for some crosses were previously published: sec2-41 sec3-2, sec5-24, sec6-4, and sec10-2 (Nair et al. 1990); sec4-8 sec3-2, sec5-24, sec10-2, and sec15-1 (Salminen and Novick 1987); sec6-4 sec1-1 and sec3-2 (Potenza et al. 1992); sec8-9 sec1-1, sec3-2, sec6-24, and sec10-2 (Bowser et al. 1992); sec15-1 sec3-2, sec5-24, and sec10-2 (Salminen and Novick 1987); sec2-41 sec15-1 (Salminen and Novick 1987; Nair et al. 1990); sec2-41 sec8-9 (Nair et al. 1990; Bowser et al. 1992); sec6-4 sec8-9 (Bowser et al. 1992; Potenza et al. 1992); sec8-9 sec15-1 (Salminen and Novick 1987; Bowser et al. 1992). stein 1985), most particularly in the requirement for a functional actin cytoskeleton to properly localize sev- eral of the post-golgi SEC gene products [Sec4p (Ayscough et al. 1997; Walch-Solimena et al. 1997) and Sec8p (Ayscough et al. 1997; Finger et al. 1998)] and for correctly polarized secretion (Novick and Botstein 1985). We find that none of the post-golgi sec mutations interacts with the act1-1 ts actin mutant allele (Figure 6). Another cytoskeletal protein, the type V myosin slow growing (Segev and Botstein 1987). The remaining allele, ypt1-2, has no growth phenotype, but is synthetically lethal in combination with some ER-to- Golgi sec mutations and is defective in an in vitro ER to Golgi transport assay (Bacon et al. 1989). We find that the post-golgi sec mutations display negative genetic interactions with all of these ypt1 mutations, although not all combinations are deleterious or lethal (Figure 5). Interaction of post-golgi sec mutations with cytoskeletal and cdc mutations: The actin cytoskeleton has been implicated in post-golgi secretion (Novick and Bot- Figure 2. Crosses of post-golgi sec mutants to mutants in SNAREs and their regulators. White boxes indicate no interaction, black boxes indicate synthetic lethality at 25, and gray boxes indicate that the restrictive temperature of the double mutant was at least 3 lower than that of either single mutant. AL, the mutations are allelic; ND, not determined. Data for some crosses were previously published: sec2-41 sec1-1, sec9-4, sec17-1, and sec18-1 (Nair 1990, no. 27); sec4-8 sec17-1 and sec18-1 (Salminen and Novick 1987); sec6-4 sec18-1 (Potenza et al. 1992); sec8-9 sec1-1 and sec9-4 (Bowser et al. 1992). Figure 3. Crosses of post-golgi sec mutants to mutants of sec4 and its accessories. White boxes indicate no interaction, black boxes indicate synthetic lethality at 25, and gray boxes indicate that the restrictive temperature of the double mutant was at least 3 lower than that of either single mutant. AL, the mutations are allelic. Data for some crosses were previously published: sec2-41 sec1-1, sec3-2, sec5-24, sec6-4, sec9-4, sec10-2, and sec19-1 (Nair et al. 1990); sec4-8 sec3-2, sec5-24, sec8-9, sec10-2, sec15-1, and sec19-1 (Salminen and Novick 1987); sec19-1 sec6-4 (Potenza et al. 1992); sec19-1 sec8-9 (Bowser et al. 1992); sec19-1 sec15-1 (Salminen and Novick 1987); dss4 sec1-1, sec2-41, sec3-2, sec4-8, sec5-24, sec6-4, sec8-9, sec9-4, sec10-2, and sec15-1 (Moya et al. 1993).

5 Synthetic Lethality of Late sec Mutants 947 Myo2p, has also been associated with post-golgi secre- with selected mutations affecting the actin cytoskeleton, tion (Johnston et al. 1991; Govindan et al. 1995), and and with selected cdc mutations affecting processes previous studies have identified a subset of post-golgi thought to be important for or involving secretion, such sec mutations (sec 4-8, sec5-24, sec8-9, sec9-4, sec10-2, and as polarity establishment and cytokinesis. As this is the sec15-1) that are synthetically lethal with myo2-66 (Govin- largest study of this kind of which we are aware, the dan et al. 1995). We have now documented an additional results are informative for interpreting synthetic lethal- synthetic lethal interaction of myo2-66 with the ity in general, in addition to the specific implications severe sec3-4 mutation (Figure 6). of these results with regard to post-golgi secretion. We were also curious as to the possibility of genetic Two models have been invoked to explain observed interaction of the post-golgi sec mutations with septins, synthetic lethal interactions of partially functional muta- as both sets of gene products are implicated in cytokine- tions (Guarente 1993), such as the ts post-golgi sec sis (Longtine et al. 1996; Finger and Novick 1997; mutations. The first model, the pipeline model, pro- Finger et al. 1998; Roth et al. 1998), and recent reports poses that reduced flux at multiple stages of a pathway suggest that septins may be localized to synaptic vesicles limits the flow through the pathway below levels re- (Beites et al. 1999) as well as to the plasma membrane quired for viability. Alternatively, synthetic lethality is and that they coassociate with members of the mammalian interpreted as resulting from genes affecting the same sec6/8 complex (Hsu et al. 1998) and with the stage of a pathway, as when mutations weaken the inter- t-snare syntaxin-1 (Beites et al. 1999). The only interactions actions of subunits of a complex, although a physical we see with cdc12-6, a tight ts allele of an essential interaction is not necessarily implied. septin (Haarer and Pringle 1987; Longtine et al. There is ample evidence, including the data pre- 1996), are synthetic lethality with sec9-4 and a weak inter- sented here, that the pipeline model does not hold true action with sec2-41 at 30 (Figure 6). for the secretory pathway, as mutations affecting ER- CDC42 and CDC24 encode, respectively, a rho-family to-golgi and intra-golgi transport are not, in general, GTPase and its GEF, which are important for establishment synthetically lethal with the post-golgi sec mutations. In of yeast cell polarity (Sloat and Pringle 1978; fact, it has previously been shown that even within the Adams et al. 1990; Johnson and Pringle 1990; Zheng ER-to-Golgi transport stage, interactions occur among et al. 1994). We have also crossed all of the post-golgi those mutations affected in vesicle budding from the sec mutants to the ts cdc24-4 and cdc42-1 mutants. We ER, or among mutations affecting fusion of ER-derived find no interaction of cdc24-4 with any of the post-golgi vesicles with the Golgi, but not between the two classes sec genes. In contrast, several of the post-golgi sec genes of mutations (Kaiser and Schekman 1990). The inter- interact with cdc42-1. Strong interactions are seen with actions of the post-golgi sec mutations with each other in sec5-24, sec8-9, sec10-2, and sec15-1, all components of almost every possible pairwise combination also indicate the plasma membrane-associated tethering complex that synthetic lethal interactions are characteristic of (Figure 6). Weaker interactions are seen with sec3-2, mutations affecting the same stage of a biological pathsec4-8, and sec9-4 (Figure 6). way. Furthermore, these extensive interactions suggest The final group of crosses we performed with the that post-golgi secretion requires the concerted action post-golgi sec mutants are those with the ts cdc28-1 mu- of all of the post-golgi SEC gene products, and that any tant. CDC28 encodes the yeast cyclin-dependent kinase, division of post-golgi secretion into substages may be an and its association with different cyclins triggers changes artificial construct, rather than a reflection of discrete in the site of secretion over the course of the cell cycle targeting, docking, and fusion steps. Although many (Lew and Reed 1993). A strong interaction between of the interactions found are between components of cdc28-1 and sec3-2, where the temperature at which lethality complexes (e.g., between sec8 and all other components occurred for the double mutant was lowered from of the tethering complex), in many cases interactions 37 in each of the single mutants to 34, with significantly were seen between genes encoding proteins not thought impaired growth at 30, was the only interaction de- to be binding partners (e.g., sec4 and ypt1). In some tected (Figure 6). cases, mutations affecting proteins that are known to physically interact did not genetically interact. For example, DISCUSSION sec17-1 and sec18-1 did not interact with mutations affecting the t-snare Sec9p or the SNARE-interacting Synthetic lethality, a strong genetic interaction protein Sec1p, even though Sec17p and Sec18p act to whereby two viable mutations are lethal when combined disassemble the SNARE complex that contains Sec9p in a double mutant, is usually interpreted as indicating (Brennwald et al. 1994; Grote and Novick 1999) and that the gene products in question function at the same that is bound by Sec1p (Carr et al. 1999). Since sec17-1 stage of a biological pathway or in parallel pathways. and sec18-1 block many stages of membrane traffic (Gra- We have completed a large study of the synthetic lethal ham and Emr 1991), it is possible that post-golgi traffic interactions of post-golgi sec mutations in combination is affected less severely than other stages at intermediate with each other, with other secretory pathway mutations, temperatures. Alternatively, the lack of interactions may

6 948 F. P. Finger and P. Novick Figure 4. Crosses of post-golgi sec mutants to early secretory pathway mutants. White boxes indicate no interaction and the gray box indicates that the restrictive temperature of the double mutant was at least 3 lower than that of either single mutant. Synthetic lethality was not observed in any of these crosses. ND, not determined. Data for some crosses were previously published: sec2-41 sec7-1, sec12-1, sec13-1, sec14-3, sec20-1, sec21-1, sec22-3, and sec23-1 (Nair et al. 1990); sec4-8 sec7-1, sec12-1, sec13-1, sec14-3, sec16-2, sec20-1, sec21-1, sec22-3, and sec23-1 (Salminen and Novick 1987); sec6-4 sec7-1, sec12-1, sec13-1, sec14-3, sec16-2, sec20-1, sec21-1, and sec23-1 (Potenza et al. 1992); sec8-9 sec7-1, sec12-1, sec13-1, sec16-2, sec20-1, sec21-1, sec22-3, and sec23-1 (Bowser et al. 1992); sec15-1 sec22-3 (Salminen and Novick 1987). indicate that the different mutations affect different aspects of SNARE complex function. While sec18-1 is known to block SNARE complex disassembly, sec1-1, sec1-13, and sec9-4 may affect complex assembly or membrane fusion. A frequently stated caution with regard to interpretation of these types of negative interactions is that the combination of two sickly mutations may result in a more severe phenotype that is additive, rather than synergistic (Botstein et al. 1997). We do not find that this is the case in our studies. For example, sec3-4, sec3-5, and sec1-13 are all quite slow growing at 25 and also display lethality in combination with the severe ypt1-1 mutation. However, synthetic lethal interactions are also seen with weaker alleles of sec1, sec3, and ypt1, suggesting that these interactions are, in fact, specific. Furthermore, none of the severe post-golgi mutations displays any synthetic phenotype with ER-to-Golgi sec mutations, or with the sickly act1-1 mutation. If the phenotypes were simply additive, we would expect to see lethality in all cases. As we do not, we see no reason to interpret any of these interactions as nonspecifically additive. In the case of the interactions of the post-golgi sec Figure 6. Crosses of post-golgi sec mutants to cytoskeletal and cdc mutants. White boxes indicate no interaction, black boxes indicate synthetic lethality at 25, and gray boxes indicate that the restrictive temperature of the double mutant was Figure 5. Crosses of post-golgi sec mutants to ypt1 alleles. at least 3 lower than that of either single mutant. ND, not ypt1 ts refers to ypt1 I121,V161 (Schmitt et al. 1988). White boxes determined. Data for some crosses were previously published: indicate no interaction, black boxes indicate synthetic lethality act1-1 sec1-1, sec2-41, sec4-8, sec6-4, sec9-4, sec10-2, andsec15-1 at 25, and gray boxes indicate that the restrictive temperature (Salminen and Novick 1987); myo2-66 sec1-1, sec2-41, sec3-2, of the double mutant was at least 3 lower than that of either sec4-8, sec5-24, sec6-4, sec8-9, sec9-4, sec10-2, and sec15-1 (Govin- single mutant. ND, not determined. dan et al. 1995).

7 Synthetic Lethality of Late sec Mutants 949 mutations with alleles of ypt1, we propose, using the Golgi vesicles upon shift to the restrictive temperature, stage-specificity model for synthetic lethality, that these suggesting that its role in secretion may be indirect interactions are reflective of a role for Ypt1p in post- (Finger and Novick 1997). Tropomyosin-containing Golgi secretion, in addition to its previously documented actin cables were recently demonstrated to be required roles in ER-to-Golgi and intra-golgi transport for the Myo2p-dependent polarized delivery of postactin (Bacon et al. 1989; Jedd et al. 1995). It has been shown Golgi secretory vesicles (Pruyne et al. 1998), and actin that the post-golgi sec mutants sec1-1 and sec6-4 accumu- is also required for polarized distribution of Sec4p (Ayscough late multiple classes of vesicles, including those that et al. 1997; Walch-Solimena et al. 1997) and are immunoreactive with antibodies against Sec4p and Sec8p (Ayscough et al. 1997; Finger et al. 1998). It is those that are immunoreactive with antibodies against possible, although unlikely given the phenotype of the Ypt1p (Mulholland et al. 1997). sec4-8 mutants also mutant, that the actin allele that was used in this study accumulate vesicles that are immunoreactive with antibodies is preferentially defective in other actin-requiring pro- against Ypt1p (Mulholland et al. 1997). These cesses. An interpretation of the lack of genetic interac- results support the suggestion that the genetic interactions tion between actin and the post-golgi sec mutants that between post-golgi sec mutations and ypt1 muta- fits the available data is that the stage of the secretory tions are indicative of the cooperation of their gene pathway at which actin functions is upstream of that products in vivo to effect exocytosis. We have previously defined by the post-golgi sec mutants. reported that high-copy SEC3 lowers the restrictive temperature Finally, the crosses with cell cycle (cdc) mutants divide for ypt1-3 and ypt1 I121,V161 (Finger and Novick the secretory mutants into several classes, in contrast to 1997). As sec3-4 and sec3-5 mutants accumulate membranes the other categories where most or all sec mutations from earlier stages of the secretory pathway, in displayed similar interactions. With the septin mutation addition to post-golgi secretory vesicles, and are partially cdc12-6, we see interactions only with sec9-4, the yeast blocked in early stages of transport (Finger and SNAP-25 homolog (Brennwald et al. 1994), and with Novick 1997), the genetic interactions of post-golgi sec sec2-41, defective in the GEF for the rab-family GTPase genes with ypt1 mutant alleles may reflect a possible role Sec4p (Walch-Solimena et al. 1997). The mammalian for Sec3p in these earlier stages of transport. septins CDCrel-1 and Nedd5 are both able to bind to The only other strong interaction seen with a muta- the t-snare syntaxin-1 (Beites et al. 1999), suggesting tion in an earlier stage of the secretory pathway is that that the genetic interaction between cdc12-6 and sec9-4 between sec14-3, defective in a phosphotidyl inositol/ could potentially reflect physical interactions of yeast phosphotidyl choline (PI/PC) transfer protein (Bankaitis septins and t-snares. A significant fraction of the mamwhen et al. 1990), and sec15-1. Sec15p is found (at least malian septin CDCrel-1 is localized to synaptic vesicles overexpressed) on post-golgi vesicles and appears (Beites et al. 1999); however, there is currently no evi- to be an effector for the rab protein Sec4p (Guo et al. dence for yeast septins localizing to post-golgi secretory 1999). If Sec15p binds to vesicles via interaction with vesicles, where Sec2p is found (Walch-Solimena et al. PI lipids, this could explain that genetic interaction. In 1997). This interaction could also indicate a role for mammalian cells an effector for Rab5, EEA1, also binds Sec2p outside of its known function in the secretory to phosphatidylinositol trisphosphate (Simonsen et al. pathway. Interactions are, somewhat surprisingly, not 1998). This could point to a general requirement for PI seen with members of the plasma membrane-associated lipids in rab effector activity. Alternatively, as both act1 vesicle tethering complex, although the corresponding and sec14-1 mutations are suppressed by sac1 (Cleves mammalian sec6/8 complex copurifies with septins et al. 1989), this interaction could also reflect the re- (Hsu et al. 1998). These results are consistent, however, quirement for actin in yeast exocytosis. with the finding that SEC gene products do not appear Although actin mutants accumulate post-golgi secre- to colocalize with septins at cytokinesis and that at least tory vesicles and are partially defective in exocytosis of one of them, Sec3p, can localize to the division site invertase (Novick and Botstein 1985), the role of actin independent of septin function (Finger et al. 1998). in secretion has been elusive. None of the post-golgi These results imply that in yeast the functions of this sec mutations interact with the ts actin mutation, act1-1 complex and the septins are separable. (this study and Salminen and Novick 1987), but such The other cdc mutations used in this study are cdc28-1, interactions may certainly be allele-specific. Mutations affecting a cyclin-dependent kinase that regulates in three actin-associated proteins, Myo2p (Govindan et changes in the sites of secretion during the cell cycle al. 1995), Pfy1p (profilin; Haarer et al. 1996; Finger (Lew and Reed 1993), and cdc24-4 and cdc42-1, affecting and Novick 1997), and Tpm1p/Tpm2p (tropomyosin; the GEF for a rho-family GTPase involved in cell polarity Liu and Bretscher 1992), do genetically interact with establishment (including establishment of the polarity the post-golgi sec mutations, and it is difficult to imagine of the actin cytoskeleton; Zheng et al. 1994) and the that such interactions would be reflective of roles for rho-family GTPase ( Johnson and Pringle 1990), respectively. these three proteins that are completely independent of We find that cdc28-1 interacts only with sec3-2, their well-characterized roles in the actin cytoskeleton. A a mild allele of SEC3. This is consistent with results that ts profilin mutant, pfy1-111, did not accumulate post- localization of Sec3p, which is important for establishing

8 950 F. P. Finger and P. Novick sites of polarized secretion in yeast, is defective only in Bender, A., and J. Pringle, 1991 Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes cdc28-1 mutants among many mutants tested (Finger involved in morphogenesis in Saccharomyces cerevisiae. Mol. et al. 1998). Interestingly, Sec3p contains one or more Cell. Biol. 11: consensus sites for Cdc28p-dependent phosphorylation Botstein, D., D. Amberg, J. Mulholland, T. Huffaker, A. Adams et al., 1997 The yeast cytoskeleton, pp in The Molecular (Finger and Novick 1998). None of the post-golgi sec and Cellular Biology of the Yeast Saccharomyces, edited by J. R. Pringle, J. R. Broach and E. W. Jones. Cold Spring Harbor Laboramutations interact with cdc24-4, but several interact with cdc42-1, which is formally downstream of cdc24. The tory Press, Plainview, NY. Bowser, R., H. Muller, B. Govindan and P. Novick, 1992 Sec8p strongest interactions are seen between cdc42-1 and sec5- and Sec15p are components of a plasma membrane-associated 24, sec8-9, sec10-2, and sec15-1, all components of the 19.5S particle that may function downstream of Sec4p to control plasma membrane tethering complex. Weaker interac- exocytosis. J. Cell Biol. 118: Brennwald, P., B. Kearns, K. Champion, S. Keränen, V. Bankaitis tions are seen with sec3-2, sec4-8, and sec9-4. The interac- et al., 1994 Sec9 is a SNAP-25-like component of a yeast SNARE tions with these cdc mutations are consistent with the complex that may be the effector of Sec4 function in exocytosis. polarity of the secretory pathway being established and/ Cell 79: Carr, C. M., E. Grote, M. Munson, F. M. Hughson and P. J. Novick, or regulated at different stages of the hierarchy for 1999 Sec1p binds to SNARE complexes and concentrates at establishment of the general polarity of the yeast cell. sites of secretion. J. Cell Biol. 146: Other possible interpretations of these data include Cleves, A. E., P. J. Novick and V. A. Bankaitas, 1989 Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin Cdc42p functioning in secretion beyond a role in the function. J. Cell Biol. 109: initial polarization of the secretory pathway, or the tethcombination Dobzhansky, T., 1946 Genetics of natural populations. XIII. Reering and variability in populations of Drosophila pseudoob- complex working with Cdc42p to perform other, scura. Genetics 31: nonsecretory functions. Finger, F. P., and P. Novick, 1997 Sec3p is involved in secretion Although the yeast genome is now completely se- and morphogenesis in Saccharomyces cerevisiae. Mol. Biol. Cell 8: quenced, no functions have yet been ascribed to a con Finger, F. P., and P. Novick, 1998 Spatial regulation of exocytosis: siderable fraction of the genes (Goffeau et al. 1996). lessons from yeast. J. Cell Biol. 142: Synthetic lethality is one of many tools that can be used Finger, F. P., T. E. Hughes and P. Novick, 1998 Sec3p is a spatial to understand the relationships between genes of both landmark for polarized secretion in budding yeast. Cell 92: known and unknown functions. Our results indicate Garrett, M. D., J. E. Zahner, C. M. Cheney and P. J. Novick, 1994 that synthetic lethality studies, while of considerable GDI1 encodes a GDP dissociation inhibitor that plays an essential utility, must be interpreted with some caution. A failure role in the yeast secretory pathway. EMBO J. 13: Goffeau, A., B. G. Barrell, H. Bussey, R. W. Davis, B. Dujon et to observe synthetic effects does not establish that the al., 1996 Life with 6000 genes. Science 274: gene products do not interact. Furthermore, the obsera Govindan, B., R. Bowser and P. Novick, 1995 The role of Myo2, yeast class V myosin, in vesicular transport. J. Cell Biol. 128: vation of synthetic interactions does not by itself reveal the underlying relationship between the gene products. Graham, T. R., and S. D. Emr, 1991 Compartmental organization of However, in combination with other more direct apevents Golgi-specific protein modification and vacuolar protein sorting defined in a yeast sec18 (NSF) mutant. J. Cell Biol. 114: proaches, synthetic lethality studies can provide impor tant insights into gene function. Griff, I. C., R. Schekman, J. E. Rothman and C. A. Kaiser, 1992 We thank Mary Travers for expert technical assistance. These studies The yeast SEC17 gene product is functionally equivalent to mammalian alpha-snap protein. J. Biol. Chem. 267: were supported by a National Institutes of Health training grant and Grote, E., and P. J. Novick, 1999 Promiscuity in rab-snare interacby a Miles Scholar Award to F.P.F., and by National Institutes of Health tions. Mol. Biol. Cell 10: grant GM to P.N. Guarente, L., 1993 Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet. 9: Guo, W., D. Roth, C. Walch-Solimena and P. Novick, 1999 The exocyst is an effector for Sec4p, targeting secretory vesicles to LITERATURE CITED sites of exocytosis. EMBO J. 18: Guthrie, C., and G. R. Fink, 1991 Guide to yeast genetics and Adams, A. E. M., D. I. Johnson, R. M. Longnecker, B. F. Sloat and molecular biology. Methods Enzymol. 194: J. R. Pringle, 1990 CDC42 and CDC43, two additional genes Haarer, B. K., and J. R. Pringle, 1987 Immunofluorescence localinvolved in budding and the establishment of cell polarity in the ization of the Saccharomyces cerevisiae CDC12 gene product to the yeast Saccharomyces cerevisiae. J. Cell Biol. 111: vicinity of the 10-nm filaments in the mother-bud neck. Mol. Ayscough, K. R., J. Stryker, N. Pokala, M. Sanders, P. Crews et Cell. Biol. 7: al., 1997 High rates of actin filament turnover in budding yeast Haarer, B. K., A. Corbett, Y. 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