Download Yeast Genes Required for Transport from the Endoplasmic

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
3406
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.
VOL. 10, 1990
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
3408
MOL. CELL. BIOL.
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
+
+
+
+
+
+
+
+
+
_
+
+
3410
MOL. CELL. BIOL.
NEWMAN ET AL.
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
3411
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.
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