MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3405-3414

Vol. 10, No. 7

0270-7306/90/073405-10$02.00/0 Copyright © 1990, American Society for Microbiology

BET], BOSI, and SEC22 Are Members of a Group of Interacting Yeast Genes Required for Transport from the Endoplasmic Reticulum to the Golgi Complex ANNA P. NEWMAN, JOSEPH SHIM, AND SUSAN FERRO-NOVICK* Department of Cell Biology, Yale University School of Medicine, Sterling Hall of Medicine, 333 Cedar Street, New Haven, Connecticut 06510-8002 Received 29 December 1989/Accepted 3 April 1990

A subset of the genes required for transport from the endoplasmic reticulum (ER) to the Golgi complex in Saccharomyces cerevisiae was found to interact genetically. While screening a yeast genomic library for genes complementing the ER-accumulating mutant bet) (A. Newman and S. Ferro-Novick, J. Cell Biol. 105: 1587-1594, 1987), we isolated BET] and BOSI (bet one suppressor). BOSI suppresses bet)-) in a gene dosage-dependent manner, providing greater suppression when it is introduced on a multicopy vector than when one additional copy is present. The BETI and BOSI genes are not functionally equivalent; overproduction of BOSI does not alleviate the lethality associated with disruption of BET). We also identified a pattern of genetic interactions among these genes and another gene implicated in transport from the ER to the Golgi complex: SEC22. Overproduction of either BET) or BOSI suppresses the growth and secretory defects of the sec22-3 mutant over a wide range of temperatures. Further evidence for genetic interaction was provided by the finding that a bet) sec22 double mutant is inviable. Another mutant which is blocked in transport from the ER to the Golgi complex, sec21-4, demonstrates a more limited ability to be suppressed by the BET) gene. The interactions we observed are specific for genes required for transport from the ER to the Golgi complex. The products of the genes involved are likely to have a direct role in transport, as bet)-) and sec22-3 begin to display their mutant phenotypes within 5 min of a shift to the restrictive temperature.

The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex involves several events that are likely to be regulated by resident intracellular proteins. In order for transport to occur, vesicles must bud from the ER and subsequently fuse with the Golgi complex (for a review, see reference 21). The dependence of intracellular membrane fusion on the presence of specific proteins has been directly demonstrated in the case of viral spike glycoproteins and postulated for fusion events which occur during endocytosis and exocytosis (for a review, see reference 39). The sorting of proteins destined to remain in the ER from those which continue to traverse the secretory pathway is mediated at least in part by protein-protein interactions (for reviews, see references 28 and 29). For instance, certain misfolded and unassembled proteins are specifically retained in this organelle in association with the binding protein BiP (5, 11, 13, 18). Many components of the cellular apparatus which participate in the sorting of proteins in the ER and in the transport of secretory proteins to the Golgi complex have yet to be identified. The finding that the secretory pathway in the yeast Saccharomyces cerevisiae is similar to that of higher eucaryotes (24) opened the way for a detailed genetic analysis of the process of protein secretion. At least 26 genes are required for the transport of proteins from the ER to the plasma membrane (3, 23, 25, 34, 36). While the function of each individual gene product must be understood at the molecular level, it is also important to elucidate the ways in which groups of these genes might interact to execute a common function. It has become clear that the power of S. cerevisiae as a genetically tractable organism resides in the ability it affords *

investigators not only to identify genes through mutational analysis but to unravel relationships among these genes as well. For instance, the finding that either of the two ct-tubulin genes in S. cerevisiae is dispensable if the other gene is present at a high enough copy number leads to the conclusion that both genes are capable of performing the same function (33). Other types of genetic interactions may be explicable in different ways. Recently, the SAC6 gene, a dominant suppressor of actin (1), was found to encode a product independently identified as an actin-binding protein (2, 12). Examples such as these make it clear that interactions that are initially understood at the genetic level may ultimately lead to enhanced biochemical insight into fundamental cell biological processes. Furthermore, they illustrate the value not only of uncovering patterns of genetic interaction but also of defining their precise nature. This may lead to testable hypotheses concerning the underlying molecular mechanism. Mutational analysis has defined 11 complementation groups of mutants which are defective in transport from the ER to the Golgi complex in S. cerevisiae (23, 25). These conditional lethal mutants have been referred to as ER accumulating based on the observation that this organelle is exaggerated at the restrictive growth temperature (37°C). Nine of these complementation groups define the ER-accumulating sec mutants, and were identified in a screen whose enrichment relied on the observation that secretory mutants become denser than the wild type (25). Subsequently, the use of [3H]mannose suicide selection resulted in the identification of two additional complementation groups of mutants defective in transport from the ER to the Golgi complex: bet] and bet2 (blocked early in transport [23]). All ER-accumulating bet and sec mutants are phenotypically similar. In the process of screening a yeast library for genes

Corresponding author. 3405

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NEWMAN ET AL.

that complement one of these mutants (beti), we isolated two genes: BET] and BOSI (bet one suppressor). In this report we document the pattern of genetic interactions that exists among BET], BOSI, and an additional gene required for transport from the ER to the Golgi complex: SEC22. The implications of these interactions are discussed. MATERIALS AND METHODS Yeast genetic methods. The yeast strains used in these experiments are listed in Table 1. These strains and their derivatives were grown in YPD medium (1% yeast extract [Difco Laboratories, Detroit, Mich.], 2% Bacto-Peptone [Difco], 2% glucose) or in minimal medium (41) with 2% glucose and appropriate amino acid supplements (37). Genetic crosses were performed essentially as described by Sherman et al. (37). Sporulated diploids were incubated in medium containing 1 M sorbitol, 50 mM potassium phosphate (pH 7.5), and 0.05 mg of zymolyase (Miles Scientific, Div. Miles Laboratories, Inc., Naperville, Ill.) per ml for 10 min at room temperature prior to tetrad dissection. To clone the BET] gene, a betl-] mutant strain was transformed with a plasmid library containing inserts of wild-type yeast genomic DNA ligated into the BamHI site of YCp5O (30). YCp5O is a shuttle vector containing a yeast centromere (CEN4) and selectable marker (URA3), as well as bacterial selectable markers (Ampr Tetr) and origins of replication (20). Transformants were tested for their ability to grow on YPD plates at 37°C. Complementing plasmids were recovered from yeast cells by the method of Holm et al. (17) and were amplified and subcloned as described below. Yeast cells were made competent for transformation by treatment with the alkali metal ion Li', as described by Ito et al. (19). Transformed cells were spread onto minimal plates lacking uracil and were grown at 25°C. To test the degree of complementation or suppression conferred by a derivative of the single-copy vector YCp5O, transformed and parental mutant strains, as well as the wild-type strain, were streaked to single colonies on YPD plates and incubated at the indicated temperatures. Colonies were scored for growth after 2, 3, and 4 days. The suppression conferred by a 2,um plasmid derivative was tested by essentially the same protocol. However, in order to select for the presence of the 2,um plasmid, transformed strains were grown on minimal plates without uracil, whereas parent strains were grown on separate plates supplemented with uracil. Nucleic acid techniques. To amplify plasmid DNA, recoinbinant plasmids were transformed into Escherichia coli DH1 (F- recAl endAl gyrA96 thi-J hsdRJ7 supE44 redAI lambda-). Plasmids were isolated from E. coli as described by Maniatis et al. (22). The plasmids used in this study (Fig. 1 and 2) were constructed as follows. (i) The plasmid pAN101 containing the BET] gene resulted from digestion of a 15-kilobase (kb) isolate complementing beti-J with HindIII and subsequent religation. To construct pAN102, the YCp5O vector was first digested with BamHI. The recessed ends were filled in with E. coli DNA polymerase I Klenow fragment (Boehringer Manheim Biochemicals, Indianapolis, Ind.), and the,linearized vector was digested with HindIlI. A 2.4-kb HindIIIPvuII fragment of yeast genomic DNA obtained from pAN101 was ligated into the resulting site. This construction preserves the HindIll site of the complementing insert, but destroys the PvuII site. pAN108 was constructed in an identical fashion, except that the YIp5 integrating vector

MOL. CELL. BIOL. TABLE 1. Yeast strains used in this study Genotype ANYll ...... MATa ura3-52 betlANY114 ....... MATa his4-619 betlANY115 ...... MATa ura3-52 his4-619 betl-J ANY119 ...... MATa ura3-52 his4-619 bet2-1 ANY122 ...... MATa ura3-52 bet2-1 ANY123 ...... MATa ura3-52 his4-619 betlANY127 ...... MATa ura3-52 his4-619 betl- (pFN100) SFNY26-4C ...... MATa ura3-52 his4-619 SFNY40 ...... MATa ura3-52 his4-619 BETJ-URA3BETJa (pAN108) SFNY52 ....... MATa his4-619 sec22-3 SFNY53 .......MMATa his4-619 sec22-3 SFNY59 ....... MATa ura3-52 (pCGS40) SFNY62 ...... MATa ura3-52 sec22-3 (pCGS40) SFNY63 ...... MATa ura3-52 sec22-3 (pFN100) SFNY64 ...... MATa ura3-52 sec22-3 (pAN109) MATa ura3-52 betl- (pCGS40) SFNY65 ...... MATa ura3-52 beti-i (pAN109) SFNY66 ...... NY3b.......MATa ura3-52 secl-1 NY13 ....... MATa ura3-52 NY15 ...... MATa ura3-52 his4-619 NY17 ...... MATa ura3-52 sec64 NY22 ...... MATa ura3-52 secS-24 NY29 ...... MATa ura3-52 sec4-8 NY44 ...... MATa ura3-52 sec8-9 NY45 ...... MATh ura3-52 sec3-2 NY57 ...... MATa ura3-52 sec94 NY61 ...... MATa ura3-52 seciO-2 NY64 ...... MATa ura3-52 seclS-I MATa ura3-52 sec241 NY130 ...... NY176 ...... MATa ura3-52 sec7-1 NY413 ...... MATa ura3-52 secl3-1 NY414 ...... MATa ura3-52 secl3-1 NY415 ...... MATa ura3-52 seci6-2 NY416 ...... MATa ura3-52 seci6-2 NY417 ...... MATa ura3-52 seci7-1 NY418 ...... MATa ura3-52 seci7-1 NY421 ...... MATa ura3-52 sec2O-J NY422 ...... MATa ura3-52 sec2O-l NY423 ...... MATa ura3-52 sec2i-J NY424 ...... MATa ura3-52 sec21-i NY425 ...... MATo ura3-52 sec22-3 NY426 ...... MATa ura3-52 sec22-3 NY427 ...... MATa ura3-52 leu2-3,112 trpi his4 secI24 NY428 ...... MATa ura3-52 leu2-3,113 his3 sec23-1 NY429 ...... MATa ura3-52 seci4-3 NY431 ...... MATa ura3-52 secl8-1 NY432 ...... MATa ura3-52 secl8-1 NY648 .MATa/a leu2-3,1121leu2-3,112 ura3-52/ ura3-52 NY737 ...... MATa ura3-52 leu2-3,112 sec23-1 NY738 ...... MATa ura3-52 seci24 a A duplication of the BET] gene marked by an insertion of URA3. Strain

b

NY strains were obtained from P. Novick.

(40), rather than YCp5O, was used. The same sites were also used in the construction of pFN100; in this case the vector pCGS40 was used (14). The HindIII-ClaI insert in pAN107 was derived from pAN102 and was cloned into the ClaI and HindIll sites of YCp5O. pAN106 was constructed by ligation of the 2.4-kb BglII fragment from pAN101 into the BamHI site of YCp5O. To construct pAN110, a 3-kb BglII fragment containing LEU2 was excised from YEp13 (8) and ligated into the unique BglII site in pFN100. To perform the gene disruption experiments, the linear fragment used was delimited by the unique HindlIl site in pAN110 and the DraI site 0.6 kb to the right of the BglII site unique to this plasmid.

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INTERACTING GENES REQUIRED FOR YEAST TRANSPORT

(ii) The BOSI clones were constructed as follows. The plasmids pAN105 and pAN109 resulted from digestion of a 15-kb isolate containing BOSI with SalI. A 6-kb fragment, extending from the Sall site shown in Fig. 2A to the SalI site in YCp5O was ligated into the SalI site of YCp5O (for pAN105) or pCGS40 (for pAN109). pAN105 and pAN109 each contain a duplication of the 0.28-kb BamHI-SalI vector fragment. As a result, the insert in each of these plasmids contains a proximal vector SphI site on both sides. To construct pFN8 and pFN9, pAN105 was digested with SphI. Two of the resulting fragments were cloned into the SphI site of YCp5O. One contained the 4.2 kb to the right of the SphI site within the cloned insert (pFN8), and the other contained the 2 kb to the left of this site (pFN9). pFN10 is a subclone of pFN8 and consists of a 3.3-kb BglII-SphI fragment ligated into the BamHI and SphI sites of YCp5O. pFN11 is a further subclone of pFN10. The recessed end of the NcoI site was filled in with the E. coli DNA polymerase I Klenow fragment, and the site was therefore not preserved in the plasmid that was constructed. The insert was ligated into the BamHI and NruI sites of YCp5O. To construct pFN13, the insert shown in Fig. 2F was treated with the E. coli DNA polymerase I Klenow fragment to fill in the recessed ends. This fragment was then blunt-end ligated into a YCp5O vector which had been digested with HindIII and filled in with the E. coli DNA polymerase I Kienow fragment. Enzyme assays. In experiments in which invertase secretion was measured, cells were suspended to an initial optical density of 1 per ml at 599 nm. At the appropriate time points, a 1-ml portion of the culture was removed and placed on ice. Cells were washed in 1 ml of ice-cold 10 mM NaN3 and suspended in 1 ml of the same solution. Internal and external invertase was then assayed as described previously (23) by the method of Goldstein and Lampen (15). RESULTS Two genes are capable of complementing the bet)-1 growth defect. To clone a wild-type copy of the BET) gene, the bet)-) mutant strain was transformed with a library of genomic yeast inserts cloned into the YCp5O vector (30). This vector is maintained at one copy per cell because of the presence of a yeast centromere and an autonomously replicating sequence. Of 47,000 transformants that were obtained, 24 demonstrated significant growth at 37°C. Plasmids isolated from these 24 strains fell into two classes. Eighteen plasmids conferred wild-type growth on the bet)-) mutant at 37°C, while the remaining six plasmids rescued the growth defect fully at 36°C but only partially at 37°C. Restriction analysis of one member of the first class of plasmids defined a 2.4-kb fragment which provided full complementation of the bet)-) mutant defect (Fig. 1B). Deletion of the 0.8 kb to the right of the ClaI site closest to NcoI (Fig. 1C) resulted in a loss of complementing activity. Similarly, deletion of the region to the left of the BgIII site (Fig. 1D) abolished the complementing activity of the clone. This therefore defined the 0.8-kb fragment extending from BglII to the most proximal ClaI site as internal to the functional complementing region. Recently, DNA sequence analysis has demonstrated that there is only one gene in this region and that it is fully contained within the 2.4-kb clone. This gene contains an intron, but does not possess a perfect 5' splice site; the precise location at which it is spliced has not yet been determined (A. P. Newman and S. FerroNovick, unpublished data). All 18 members of the first class of plasmids were found to contain the 0.8-kb BglII-ClaI

3407

fragment (data not shown). To determine whether the 2.4-kb complementing insert contained the BET) structural gene, this fragment was ligated into the yeast integrating vector YIp5 (40). This vector does not contain the elements necessary for autonomous replication in S. cerevisiae, and thus, it must integrate into the yeast genome in order to be maintained. The resulting plasmid (Fig. 1B) was cut within the 2.4-kb complementing insert to stimulate recombination with the homologous region in the yeast genome and was used to transform the wild-type yeast strain SFNY26-4C (ura3-52 his4-619). This generated a strain containing URA3 adjacent to the locus of the cloned sequence. This transformed strain was then crossed to a ura3-52 bet)-) mutant strain, and the resulting diploids were sporulated and dissected. The Ura+ and Ts' phenotypes cosegregated in all 19 tetrads analyzed, indicating that the locus we identified was tightly linked to the BET) gene. Restriction analysis of one member of the second class of plasmids (Fig. 2) revealed it to be distinct from the region containing the BET) gene. Subcloning experiments were used to define the region responsible for suppressing bet)-) (Fig. 2A through F). A 2.2-kb NcoI-HincII fragment (Fig. 2F) was found to provide the same degree of suppression as the initial isolate. In evaluating the ability of BOSI to suppress other mutants blocked in transport (see below), a 5.8-kb insert (Fig. 2A) was used which extended beyond this fully suppressing region by 2.2 kb to the left and 1.4 kb to the right. It therefore seemed unlikely that this 5.8-kb fragment contained a truncation of the BOSI gene. Recently, DNA sequence analysis has shown this assumption to be correct (unpublished data). The five additional members of the second class of plasmids were each demonstrated by restriction analysis to contain the 2.2-kb NcoI-HincII fragment (data not shown). The gene present in the second class of plasmids was named BOSI (bet one suppressor). The bet) mutant defect was also suppressed in a strain in which a duplication of BOSI was generated by using the integrating vector YIp5. This gene duplication was confirmed by Southern blot analysis (data not shown). The suppression of bet) that we observed was most likely due to the presence of an additional copy of the BOSI wild-type gene, as six independent isolates were obtained. However, this finding does not exclude the possibility that the strain used to construct the library from which BOSI was cloned contained a spontaneous mutation in this gene. To determine whether the bet)-) growth defect could be more effectively rescued if the BOSI gene was present on a multicopy plasmid, we transformed this mutant with pAN109 (Fig. 2A). This generated the SFNY66 strain (Table 1). When growth of single colonies on a plate was compared at 37°C, SFNY66 appeared indistinguishable from the wild type. Thus, BOSI suppresses bet)-) in a gene dosagedependent manner, providing greater suppression when it is present on a multicopy plasmid than when one additional copy is present. Using antibody directed against the BOSI gene product, we have shown that an increase in the BOSI gene dosage is accompanied by substantial overproduction of the 27-kilodalton Bosl protein (unpublished data). Overproduction of BET) or BOSI specifically suppresses mutants that block transit from the ER to the Golgi complex. Does the genetic interaction between BET) and BOSI also extend to other genes that are involved in transport? To address this question, one member of each complementation group of sec and bet mutants accumulating ER, Golgi complex, or post-Golgi complex vesicles (23, 25) was transformed with either the BOSI or BET) gene on a multicopy

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NEWMAN ET AL.

Complementation ofbellA pAN101

H

B

N

D C

B pAN102, 108; pFN100

p

C D

B

D

Ba/Sa

+

D

+

C pAN107

D pAN106

E pANllO

B Leu

FIG. 1. Complementing activity of clones containing the BET] gene. Only the cloned insert, and not the vector portion of each plasmid, is shown. Plasmids are described in the text. The LEU2 fragment used to disrupt BET] (E) is not drawn to scale. B, BglIl; C, ClaI; D, DraI; H, HindIII; N, NcoI; P, PvuII; Ba/Sa, BamHI-Sau3A junction; bp, base pairs.

plasmid. The growth of transformed strains was compared with that of parental strains at the indicated temperatures (Table 2). We found that overproduction of either BOSI or BET) was capable of suppressing the ER-accumulating mutant sec22-3 over a broad temperature range. This phenomenon does not appear to be allele specific, as the sec22-2 mutant allele is also suppressed (A. P. Newman and S. Ferro-Novick, unpublished data). Another ER-accumulating mutant, sec21-1, can be suppressed to a more limited degree by overproduction of BET]. Neither the BET) nor the BOSI gene can significantly improve the growth of mutants blocked at any other stage of transport. Thus, the genetic interactions that we observed were limited to genes involved in one aspect of the secretory process: the movement of proteins from the ER to the Golgi complex. Overproduction of the BOSI gene slows the growth of certain mutants. For instance, the Golgi complex-accumulating mutant sec7-1 normally exhibits wild-type growth at 34°C. However, transformation of this strain with BOSI results in a complete absence of growth at this temperature. Two additional mutants are affected by overproduction of BOSI in a similar manner: sec8-9 (which accumulates postGolgi complex vesicles) and secl4-3 (Golgi complex-accumulating). This is not due to an increased frequency of loss of the 2jxm plasmid in these strains, as transformation of sec7-1, sec8-9, or secl4-3 with the parent 2,um plasmid has no effect on growth (data not shown). One possible explanation is that overproduction of BOSI may have a somewhat deleterious effect even on wild-type cells and may provide

sufficient additional stress to certain mutant strains to render them inviable at normally permissive temperatures. In support of this hypothesis, we note that transformation of a wild-type strain with BOSI on a 2,um plasmid results in a slight reduction in the rate of growth. This increase in doubling time is more pronounced when a galactose-inducible promoter is used to achieve a greater level of overproduction of BOSI (data not shown). The reason that some mutants are affected more than others remains obscure. The data in Table 2 also indicate that overproduction of BOS1 does not complement the growth defect of any secretory mutant other than bet) at the completely restrictive temperature of 37°C. Together with the data from restriction analysis discussed above, this demonstrates that BOSI does not correspond to any of the SEC or BET genes previously identified as being necessary for transport through mutational analysis. The BET) and BOSI genes are not functionally equivalent. One explanation for the observation that overproduction of BOSI can suppress the bet)-) mutant defect is that the BOSI gene product is capable of performing the BET) function. To address this possibility, we first needed to assess the phenotype that would result from disrupting the BET) gene. The existence of a conditional lethal mutation in BET) suggested that the locus might be essential for cellular viability. To determine this directly by disruption of the BET) locus, we used the Bglll site that was demonstrated to be internal to the region of complementing activity. A 3-kb BglII fragment containing LEU2 was excised from YEp13 (8) and inserted

VOL. 10, 1990

INTERACTING GENES REQUIRED FOR YEAST TRANSPORT

3409

Suppression of bet]-I A pANlOS,

H

N

N

B

E

S

Ba

S

pAN109

BaISau

N

H

N

B pFN8

+

+

C 'pFN9

D pFN1O

+

E pFNIl

+

F pFN13

+

.500 FIG. 2. Ability of plasmids containing the BOSI gene to suppress betl-l. Only the cloned insert, and not the vector portion of each plasmid, is shown. Plasmids are described in the text. Ba, BamHI; B, BglII; E, EcoRI; H, HincII; N, NcoI; Sa, Sall; S, SphI; Ba/Sau, BamHI-Sau3A junction; bp, base pairs. bp

TABLE 2. Overproduction of BET) or BOSI results in stage-specific suppression of ER-accumulating mutants Growth of the following strains at the indicated temperatures: Strain

ER-accumulating ANY123 ANY119 NY427 NY413 NY416 NY417 NY432 NY421 NY424 NY425 NY737

Golgi complex-accumulating NY176 NY429

Genotype

Strain transformed with BET)

Parental strain

Strain transformed with BOSI

300C

34°C

37°C

30°C

34°C

37°C

300C

34°C

37°C

bet)-)

+ +

± _ _

-

+ + _ + _ +

+ _ -

+ + _ + + +

-

-

±

+ -

+ +

secl24 secl3-1 secl6-2 secl7-1 secl8-1 sec20-1

+ _ +

+

bet2-1

±

-

-

_ + +

+ +

±

+ + +

sec7-1 secl4-3

+ +

+ _

-

+ +

+

-

+

secl-l sec241 sec3-2

+

-

-

+

-

-

+

±

-

-

+ + +

-

-

+ + +

-

+

+

+

±

-

+ +

sec21-1 sec22-3 sec23-1

+

_ +

+

Vesicle-accumulating (postGolgi complex) NY3 NY130 NY45 NY29 NY22 NY17 NY44 NY57 NY61 NY64

+

_ +

+ +

sec4-8

secS-24 sec64 sec8-9 sec94 seclO-2 seclS-1

+ + +

+

+

+ + +

+

_

+ +

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into this site (Fig. 1E). A linear HindIII-DraI fragment containing the disrupted locus was used to transform a diploid strain homozygous for the leu2-3,112 mutations. The ends of this segment were homologous to sites within the BET] chromosomal locus, thus stimulating homologous recombination and replacement of one chromosomal copy of BETI with the disrupted allele. The transformed diploid was sporulated and dissected. Of 12 tetrads analyzed, 11 demonstrated a ratio of 2:2 viable to inviable spores, with both viable spores being Leu-. One tetrad contained three viable spores, two of which were Leu+, and was probably a false tetrad. These results demonstrate that the BET] locus is essential for cellular viability. The finding that the BET] locus is essential for cellular growth makes it clear that the chromosomal copy of BOSl is incapable of compensating for the absence of BET]. However, it was still possible that BET] and BOSI were functionally equivalent but that insufficient gene dosage was provided by one copy of BOSI. In this case, we would expect that overproduction of BOSI could alleviate the lethality associated with disruption of BET]. To test this possibility, a diploid strain which was homozygous for the ura3-52 and leu2-3,112 mutations (NY648) was first transformed with the linear HindIII-DraI fragment containing a disrupted copy of the BET1 locus. Leu+ transformants, in which a disrupted copy of BET] replaced one genomic copy of this gene, were then transformed with pAN109, a plasmid containing the BOSI and URA3 genes on a multicopy plasmid. If overproduction of BOSI were capable of rescuing the growth defect caused by disruption of BET], we would expect to be able to obtain spores which were both Ura+ and Leu+. Instead, we found that of 28 Ura+ spores obtained, all were Leu-. Thus, overproduction of BOSI, while capable of suppressing the betl-l mutant defect, cannot alleviate the lethality associated with disruption of the BET] gene. We conclude that the BET] and BOSI genes are not functionally interchangeable. The BETI and BOSI genes suppress both the growth and secretory defects of ER-accumulating mutants. To evaluate whether the suppression of growth observed was related to the block in secretion, we used wild-type and mutant (betl-l or sec22-3) strains which were transformed with a 2,um plasmid containing BET] or BOSI. Both growth rate and secretion were measured on cells grown in liquid culture. When a betl-l strain (transformed with a 2,um vector without an insert) was grown at 36°C, the cells failed to undergo even one doubling (Table 3). In contrast, a betl-l mutant containing BOSI on a multicopy plasmid grew at approximately half the rate of the wild type. As described earlier, in experiments in which growth of single colonies on a plate was compared, a betl-l strain containing BOSI on a multicopy plasmid appeared to grow as well as the wild type. However, since measurements in liquid culture are made when cells are growing logarithmically, they are probably more accurate. In any case, it is apparent that overproduction of BOSI substantially ameliorates the betl-l growth defect under all conditions studied. To measure secretion, the marker enzyme invertase was followed. Yeast cells contain two forms of invertase. Cytoplasmic invertase is constitutively synthesized, while production of the secreted form of the protein is controlled by hexose repression (9, 27). Mutants defective in transport from the ER to the Golgi complex fail to secrete the hexose-repressible form of invertase. Instead, enzymatically active precursors are retained within the ER (23, 25). To determine whether the betl-l secretory defect would be

TABLE 3. Growth and secretory defects of betl-i and sec22-3 are suppressed by overproduction of BOSI or BET]

Expt ia betl-i

bell-i with BOSI Wild type Expt 2b sec22-3 sec22-3 with BET] sec22-3 with BOSI Wild type

Doubling time (h, mm)

% Invertase

No doubling 4, 35 2, 5

19 56 98

No doubling 6, 50 7, 0 3, 0

38 61 62 100

secreted

a For the determination of growth rate and invertase secretion of betl-i in the presence or absence of BOSI on a multicopy plasmid, yeast strains (SFNY59, SFNY65, SFNY66) were grown overnight at 24°C to the early exponential phase in minimal medium containing 2% glucose. To measure the rate of growth, cells were pelleted, suspended in fresh medium, and grown at 36'C. To measure invertase secretion, cells were pelleted, suspended in medium containing 0.1% glucose, and grown at 36°C for 1 h. Invertase was then assayed as described in Materials and Methods. Percent invertase secreted was calculated as follows: [external invertase/(external invertase + internal invertase)] x 100. b For the determination of growth rate and invertase secretion of sec22-3 in the presence or absence of BET] or BOSJ on a multicopy plasmid, the protocol described above was followed, except that the strains SFNY59, SFNY62, SFNY63, and SFNY64 were used and the experiments were performed at 31'C.

affected by overproduction of BOSI, the appropriate strains were shifted to 36°C for 1 h. The amount of invertase secreted into the periplasmic space (external invertase), as well as the amount retained within the cell, was quantitated. The percentage of total invertase synthesized during 1 h which was secreted into the periplasmic space was calculated. At 36°C, only 19% of the invertase produced by the betl-l mutant was secreted, as compared with 98% in the case of the wild type (Table 3). In a bet] strain which was transformed with BOSI on a multicopy plasmid, 56% of the total invertase was secreted. This value is midway between that obtained for the betl mutant alone and that obtained for the wild type. We conclude that BOSI suppresses the growth and secretory defects of betl-l and that it suppresses both to approximately the same extent. Similar experiments were performed with a sec22-3 mutant strain that was transformed with a multicopy plasmid containing BET], BOSI, or no insert. These experiments were done at 31°C. It was found that overproduction of either gene can suppress both the growth and the secretory defects of sec22-3 (Table 3). These defects are suppressed to a somewhat lesser degree than those of betl-l are. This is consistent with another observation regarding the degree of genetic interaction observed among BETI, BOSI, and SEC22. Namely, the betl, but not the sec22, mutant can be suppressed by the presence of BOSI on a single-copy vector (data not shown). Thus, by several criteria, it seems that the interaction between BET1 and BOSI was the strongest observed in this study. It is interesting that the BET] and BOSI genes are both equally efficient at suppressing the sec22 mutant defects (Table 3). A bet] sec22 double mutant is inviable. Inviability of double mutants is another means of documenting genetic interaction. If the effect of combining two mutations is to cause lethality under normally permissive conditions, the explanation may be that the mutated genes encode products with related functions. Then, the combined effect of both mutations might be to hinder a single process to a degree that far

INTERACTING GENES REQUIRED FOR YEAST TRANSPORT

VOL. 10, 1990

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TABLE 4. Tetrad analysis of crosses between betl-] or sec22-3 and the ER-accumulating mutantsa No. of crosses Cross

betl- crossed with: bet2-1 secl24

4- :o+b

3-:1+

2-:2+

3 2

6 2 8 9 10 8 10 5

3 4 2 1

6

3

9 10 10 8 8 9 7 6 11

6 2 1 4 5 4 2 3 3

sec13-1 secl6-2 secl 7-1 secl8-1 sec20-1 sec21-1 sec22-3 sec23-1 sec22-3 crossed with: bet2-1 secl24 secl3-1 secl6-2 secl 7-1 secl8-1 sec20-1 sec21-1 sec23-1

3 3 1 2 3 1 1 3 4 3 3 1 6 3 1

3-:0+

2-:1+

One viable

Two viable

Three viable

Four viable

1-:2+

2-:0+

1-:1+

0-:2+

(O-:1+)

1

4

1

1 1 1

3

2

5 3 2

1 23

1

1 3

1

2

1 1 1

1 2

1

1

a All spores were tested for the presence of the URA3 and HIS4 selectable markers as well as for temperature sensitivity. Those tetrads for which two or more markers segregated aberrantly were considered false tetrads and are not presented here. b Designates the number of spores that grew (+) or did not grow (-) at 37°C.

exceeds the impairment caused by either mutation alone. If this process is essential for cellular viability, "synthetic lethality" (6) can result. Studies in which the observed pattern of synthetic lethality has correlated well with other methods of assessing genetic interaction (32) support the interpretation that this phenomenon is not merely due to the nonspecific effect of combining two mutations. In this study, we crossed beti-) and sec22-3 to each other and to each of the nine additional ER-accumulating mutants. When beti-) was crossed to sec22-3, the majority of tetrads had three viable spores (Table 4). This is the expected result if a double mutant is inviable. Furthermore, when the viable spores obtained were tested for their ability to complement betl-J or sec22-3, it was found that none of the spores was a beti sec22 double mutant (data not shown). In contrast, in all other crosses, the majority of tetrads had four viable spores. The fact that only the combination of betl-J and sec22-3 resulted in synthetic lethality is consistent with the conclusion that BET] and SEC22 are genetically interacting genes. Since we showed that the sec21-1 mutant could be suppressed by overproduction of BET], we examined the spores resulting from the cross between bet)-) and sec21-1 more carefully. Subsequent to incubation at 25°C, colonies were stamped onto replica plates, which were then grown at 25, 30, 34, and 37°C. Spores containing either the bet)-) or the sec21-1 mutation alone grew at 30°C, whereas spores containing both mutations failed to grow at this temperature. Since the sec22-3 mutant does not grow well at 30°C, it was not possible to analyze the spores resulting from the cross between sec22-3 and sec21-I in the same way. In general, the results of the double-mutant analysis confirm those obtained in experiments described above. The evidence strongly supports the interaction of BET] and SEC22 and suggests that SEC21 may also be involved in this interaction. The bet)-) and sec22-3 mutants act quickly upon a shift to

the restrictive temperature. If the BET) and SEC22 genes have a direct role in transport, we would expect the corresponding mutants to display their phenotypes soon after a shift to the restrictive temperature. To assess the degree of the bet)-) secretory block at early time points, we followed the enzyme invertase, using the experimental design of Salminen and Novick (32). Cells were first derepressed for the synthesis of secreted invertase at 25°C for 35 min and then shifted to the restrictive temperature of 37°C. Invertase activity inside the cell and in the periplasmic space was measured at regular intervals (Fig. 3). We found that enzymatically active invertase began to accumulate within the cell at the earliest time point measured: 5 min after the shift to 37°C. In contrast, control strains maintained at 25°C exhibited no such accumulation. A slightly longer time (10 min) elapsed before mutant cells at 37°C began to secrete significantly less invertase into the periplasmic space than did cells maintained at 25°C. This was probably due to an initial leakiness of the block directly following a shift to the restrictive temperature. In any case, it is clear that the phenotype of bet)-) first becomes evident very shortly after the shift to the restrictive temperature. When similar experiments were performed with sec22-3 cells, virtually identical results were obtained (data not shown). Thus, both bet)-) and sec22-3 strains begin to assume their mutant phenotypes extremely rapidly once they are placed at the restrictive temperature. This suggests that both the BET) and SEC22 genes have a direct role in mediating the transport of secreted proteins from the ER to the Golgi complex in S.

cerevisiae. DISCUSSION In this report, we described the identification of BOSI, a gene which provides stage-specific suppression of two genes

3412

MOL. CELL. BIOL.

NEWMAN ET AL.

A i

0.07 '

0W

0.06 -

EI-

0.05 Shift to 370C

w

0

0.04

0-

0.03

-4

0.02

0% Sn

Shift to 0.1% glucose

250C

0.01 0.00

0

E

10

20

30

40

50

60

Time (min)

B

c;P.

0.07 1-

s 0

0.06

370C

Shift to 37°C

0.05

m

xr

0.04

0.03 Shift to 0.1% glucose

0.02

0.01 0.00 0

10

20

30 Time (min)

40

50

60

FIG. 3. Secreted and intracellular invertase in the betl-l mutant. The betl-l mutant strain ANY123 was grown overnight at 24°C to the early exponential phase in YPD medium. Cells were pelleted, suspended in YP medium containing 0.1% glucose, and grown at 24°C for an additional 35 min. Cells were then shifted to 37°C. Portions were removed at the indicated times and assayed for the presence of internal (A) and external (B) invertase as described in Materials and Methods.

required for transport from the ER to the Golgi complex in S. cerevisiae. We also show that these two genes, BET] and SEC22, interact with one another as well as with BOSI. Here we discuss possible mechanisms of suppression and also consider further implications of this study. Recent experiments have shown that introduction of additional copies of the BOSI gene into yeast cells leads to overproduction of the Bosl protein (unpublished data). It therefore seems likely that this change in Bosl protein level results in the suppression of betl-J and sec22-3 that we observed. This could occur at the level of gene regulation or could be a consequence of functional interaction among the BOSI, BET], and SEC22 gene products. For instance, these proteins might exist in a physical complex with one another, as has been demonstrated in several cases involving structural proteins which interact genetically (2, 16, 38). Alternatively, the data presented in this study may reflect the fact that BOSI, BET], and SEC22 are acting on the same or a parallel pathway. The finding that the BOSI gene product

itself appears to be required for transport from the ER to the Golgi complex (see below) increases the likelihood that one of the latter two interpretations is correct. Although the precise mechanism of suppression has not been defined, the genetic interactions documented in this study have led to the identification of a new gene (BOSI) that appears to play a role in the transport of secreted proteins from the ER to the Golgi complex. Previously, mutational analyses identified 11 SEC and BET genes required for transport from the ER to the Golgi complex in S. cerevisiae (23, 25). Complementation analysis has shown BOSI to be distinct from each of these genes. The finding that BOSI interacts with both BET] and SEC22, but not with genes involved in later stages of secretion, led us to postulate that the BOSI gene product may itself be required for transport from the ER to the Golgi complex. Experiments in which a regulatable promoter was used to deplete wild-type yeast cells of the Bosl protein have provided support for this hypothesis. Specifically, precursors to transported proteins

VOL. 10, 1990

INTERACTING GENES REQUIRED FOR YEAST TRANSPORT

accumulate that are core glycosylated but that have not acquired outer-chain carbohydrate, a Golgi complex-specific modification. Thin-section electron microscopy has revealed the accumulation of ER membrane in these cells (unpublished data). Although overproduction of BOSI can suppress the betl-] mutant defect, this is not due to functional equivalence between the two genes; a strain in which the BET] gene is disrupted is inviable even when BOSI is present on a multicopy plasmid. Recently, several other instances have been reported in which duplication of a yeast gene is capable of suppressing the mutant defect of a gene with a related function. Duplication of SEC4, a gene required for post-Golgi complex secretion in S. cerevisiae, suppresses mutations in several other genes which function at the same stage of the secretory pathway (32). A similar phenomenon has been found for two genes required for bud emergence; one additional copy of CDC42 is sufficient to suppress a mutation in CDC24 (4). Several general conclusions can be drawn from these studies as well as from those in which suppression is dependent on substantial overexpression (for example, see reference 10). First, they represent one means of augmenting the number of genes that can be isolated as a consequence of classical genetic screens, since the classical approach permits identification of only those genes which can be mutated to give a temperature-sensitive or cold-sensitive allele. Second, cases such as those discussed above lead to the definition of two proteins (encoded by a given gene and its suppressor) with potentially overlapping or intersecting functions within the cell. The work presented in this report has linked three genes required for transport from the ER to the Golgi complex on the basis of their genetic interactions. This should provide a framework for future biochemical studies, which may distinguish between the various models that could explain this phenomenon. Morphological and biochemical analyses have provided evidence that protein transport between these organelles involves the budding of vesicular carriers from the ER and their subsequent fusion with the Golgi complex (26; for a recent review, see reference 21). An assay developed in this laboratory, which reconstitutes transport from the ER to the Golgi complex in vitro (31), has recently been used to isolate a vesicular transport intermediate which is capable of fusing with an acceptor (Golgi complex) compartment (M. Groesch, H. Ruohola, R. Bacon, G. Rossi, and S. Ferro-Novick, J. Cell Biol., in press). Thus, the processes of budding and fusion are distinct biochemical events which are separable in vitro. It seems likely that the interactions reported here reflect the participation of a number of proteins in one of these events, either budding or fusion. However, as the budding of vesicular carriers from the ER and their subsequent fusion with the Golgi complex are closely related phenomena, it is also conceivable that some of these gene products may function in both processes. Biochemical analysis of the Betl, Bosl, and Sec22 proteins in vivo and in vitro should enable us to determine whether they participate in budding or in fusion and to evaluate their particular functions in detail. ACKNOWLEDGMENTS We are grateful to Becky Bacon, Mary Groesch, Alisa Kabcenell, Peter Novick, and Nancy Walworth for comments on the manuscript. We thank Chris Kaiser and Randy Schekman for providing us with the sec22-2 allele. This work was supported by grants MV-422 from the American Cancer Society and Public Health Service grant CA 46128 from the

3413

National Institutes of Health (to S.F.-N.). A.P.N. was a recipient of a predoctoral fellowship from the National Science Foundation. LITERATURE CITED 1. Adams, A. E. M., and D. Botstein. 1989. Dominant suppressors of yeast actin mutations that are reciprocally suppressed. Genetics 121:675-683. 2. Adams, A. E. M., D. Botstein, and D. Drubin. 1989. A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation. Science 243:231-233. 3. Bacon, R. A., A. Salminen, H. Ruohola, P. Novick, and S. Ferro-Novick. 1989. The GTP-binding protein, Yptl, is required for transport in vitro: the Golgi apparatus is defective in yptl mutants. J. Cell Biol. 109:1015-1022. 4. Bender, A., and J. R. Pringle. 1989. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSRI. Proc. Natl. Acad. Sci. USA 86:9976-9980. 5. Bole, D. G., L. M. Hendershot, and J. F. Kearney. 1986.

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