From the VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Finland
Received for publication, December 23, 2002
, and in revised form, March 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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In a genetic screen we previously identified SEB1/SBH1 as
a multicopy suppressor of the sec15-1 exocyst mutant
(20). SEB1
(SBH1) and its homologue SEB2 (SBH2) encode the
subunits of the trimeric Sec61p and Ssh1p ER translocation complexes,
respectively (20,
21,
22). The
subunits of
the Sec61p and the Ssh1p complexes are Sec61p and Ssh1p, respectively, which
have been shown to form the protein conducting channel
(23) and the ribosome binding
site at the ER (24). The
subunit in both complexes is Sss1p
(22,
25). These complexes have been
shown to function in cotranslational translocation and the Sec61p complex also
in the posttranslational translocation
(21,
22,
26). The exact functions of
the
subunits of the ER translocation complexes are not known.
We have now extended our previous finding on the genetic link between
SEB1 and sec15-1 mutation and provide biochemical and
functional evidence for protein-protein interactions between the ER
translocation complex subunit and components of the exocyst complex and
Sec4p.
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EXPERIMENTAL PROCEDURES |
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Plasmids and DNA MethodsPlasmids YEpHA-SEB1 and pBSEB1 have
been described (20). The
SEB1 cDNA covering the open reading frame was transferred from pBSEB1
under TPI1 promoter in pYX212 multicopy vector (R&D Systems) as
an EcoRI-XhoI fragment yielding plasmid YEpT-SEB1U. YEpSEB2U
is pRS426 (29) containing the
SEB2 expression cassette as a BamHI-HindIII
fragment from YEpSEB2 (20).
YEpSEC1aU overexpressing SEC1 and YEp24H
(30) as well as YEpSSO1U and
YEpSSO2U overexpressing SSO1 and SSO2, respectively, have
been described (31). For
disruption of the URA3 gene, pBUF containing a 331-base pair (bp)
deletion within the 1135-bp-long URA3 coding region was constructed
as follows. Two URA3 fragments were amplified by PCR and cloned into
the multiple cloning site of pBluescript SK(-). The first fragment comprising
bp 71450 of the URA3 was cloned as a
SacI-XbaI fragment and the second (bp 781-1135) as a
XhoI-KpnI fragment. pBW65 containing the SEC61 gene
in YEp351 vector (32) and
FKp53 (33) containing the
SSS1 gene in YEp352 were obtained from Colin Stirling. The control
plasmids for pBW65 and FKp53 were pRS425
(29) and YEp24H
(30), respectively, and for
YEpHA-SEB1 was pVT102U (34).
To generate a His6-tagged Sec4p, the coding region was amplified by
PCR with BamHI-XhoI sites added and cloned into a version of
pGAT (provided by Johan Peränen) from which the GST coding region was
removed by SpeI digestion and religation. This resulted in
pGATHis-Sec4p. To generate a His6-tagged version of the first 241
amino acids of Sec15p, the coding region for these amino acids was amplified
by PCR and cloned as a BamHI-EcoRI fragment to pRSETA
(Invitrogen) to create pSET-His-Sec15 (amino acids 1241). The fragment
encoding Seb1p without transmembrane domain (amino acids 154) was
created by PCR with oligonucleotide primers containing XbaI and
XhoI sites and ligated into XbaI-XhoI cut pVT102U
(34) yielding YEpSEB1-MA. The
plasmid YIplac204a for
-amylase gene integration was obtained by
inserting the
-amylase expression cassette (Bacillus
amyloliquefaciens
-amylase gene between ADH1 promoter and
terminator) from YEp
a6
(35) as
BamHI-SalI fragment into the multiple cloning site of
YIplac204 (36). SEC3
in YEp352 (37) was obtained
from Brian Haarer, YEpSEC4 (NRB524) and pNB148 overexpressing SEC4
(38) and SEC15
(39), respectively, from Peter
Novick, and pRD6 and pDF11 overexpressing SEC62
(40), and SEC63
(41), respectively, from Randy
Schekman. Standard DNA methods were used for cloning. The yeast cells were
transformed as described
(20).
Multicopy SuppressionThe mutant strains were transformed with the multicopy plasmid overexpressing the gene under study or with the pertinent vector control. The multicopy suppression was first screened by replica plating freshly grown patches on selective plates and on YPD and growing them at different temperatures. Growth was monitored for 3 successive days. The suppression test was then done by dotting 105 cells and three 10-fold dilutions thereof on selective plates and incubating the plates at different temperatures for 3 days. The patching method was slightly more sensitive and rescued the growth at a slightly higher temperature or detected in some cases weak suppression that was not detected by dotting.
Determination of -Amylase and Invertase
ActivityYeast cell growth, preparation of the periplasmic and
cytoplasmic fractions, and determination of the
-amylase activity were
done as described previously
(42). Invertase activity was
determined as described (42)
except that the reaction was terminated with addition of 1 M Tris,
pH 8.5, after which the cells were pelleted instead of filtering and the
glucose liberated from sucrose by invertase was measured from the
supernatant.
Antibodies and Protein MethodsAntibodies against an N-terminal peptide of Seb1p have been described (20). The antibodies against Sec4p and Sec15p were raised in rabbits injected with bacterially (Escherichia coli BL-21de3) expressed and purified His6-Sec4p and His6-Sec15p (amino acids 1241). HA (12CA5) and Myc tag antibodies (9E10) were purchased from Roche Applied Science and Santa Cruz Biotechnology, respectively. For detection of Sec4p in Western blots, an IgG heavy chain-specific secondary antibody (Jackson ImmunoResearch Laboratories Inc.) was used to reduce signals from closely migrating IgG light chain specific bands. Other antibodies were generous gifts from Howard Riezman (antibodies against Gas1p) and Romano Serrano (anti-Pma1p antibodies). The proteins were separated in SDS-PAGE using the buffer system of either Laemmli (43) or Schägger and von Jagow (44) with acrylamide concentrations from 7.5 to 15% as appropriate for the proteins analyzed and transferred onto Hybond-P filter (Amersham Biosciences). The protein bands were visualized with the ECL detection system (Amersham Biosciences).
Preparation of Yeast Cell Lysates and
ImmunoprecipitationsTransformants of strains H2647 and NY15 were
grown overnight in SCD-Ura to early logarithmic growth phase of
A600 = 1 and pelleted. The cells were washed with 10
mM NaN3 and resuspended in spheroplasting buffer (1.4
M sorbitol, 20 mM triethanolamine, pH 7.5, 40
mM -mercapthoethanol) containing 10 mM
NaN3. Cells were converted to spheroplasts with 150 µg/ml
zymolyase (Seikagaku Corp.). The spheroplasts were cooled on ice, pelleted,
washed with spherowash (20 mM triethanolamine, pH 7.5, 1.4
M sorbitol), and gently lysed in either homogenization buffer (20
mM Hepes, pH 7.4, 100 mM NaCl 1 mM EDTA 1%
Nonidet P-40) or buffer B (20 mM Pipes, pH 6.8, 100 mM
NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40)
by repeated pipetting and 30 min of incubation on ice. Buffer B was utilized
to improve the stability of the Sec15-Sec8p complex
(45). Seb1p was coprecipitated
with
Sec15p antibodies with equal efficiency from cell lysates prepared
either in homogenization buffer or buffer B. The cell homogenates were
centrifuged at 10,000 x g for 10 min at 4 °C, and the
supernatant was used for immunoprecipitations.
1.1 mg of total protein of cell lysates in homogenization buffer or buffer B was precleared with protein G-Sepharose (Amersham Biosciences) for 30 min at 4 °C and centrifuged at 20,000 x g for 15 min. Precleared lysate containing 1 mg of total protein was subjected to overnight immunoprecipitation with 3 µl of antiserum. Immunoprecipitations were centrifuged (17,000 x g for 1 min); the supernatant was transferred to new tubes, and 20 µl of 50% protein G-Sepharose in the same buffer was added. The immunocomplexes were bound to the beads in an end-over-end mixer for 2 h at room temperature. The beads were washed five times with 1 ml of the lysis buffer and once with 1 ml of 10 mM Tris, pH 6.8. The Complete Protease inhibition mixture (Roche Applied Science) and 4 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (Roche Applied Science) were present during homogenization and preparation of the lysates and immunoprecipitations but were omitted from the washes. The immunocomplexes were released into 20 µl of Laemmli sample buffer, heated at 95 °C for 5 min, and analyzed by SDS-PAGE and Western blotting. For detection in Western blots, anti-Seb1p was used in 1:2000 to 1:8000 dilutions. Sec8p-Myc was detected with c-Myc-specific antibody (1:500). Anti-Sec4p was used in a 1:80000 dilution and IgG heavy chain-specific antibody in a 1:100000 dilution. Pma1p was detected with anti-Pma1p diluted 1:100000 and Gas1p with anti-Gas1p diluted 1:10000. The bound antibodies in Western blots were visualized with the ECL detection system (Amersham Biosciences). The quantitations were done from Kodak Biomax MR films using a GS-710 imaging densitometer and Quantity One software (Bio-Rad). The immunoprecipitates were quantitated by comparing the amount of a given protein precipitated to the amount of that protein in the cell lysate that was used for precipitation.
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RESULTS |
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We also studied possible synthetic interactions between
seb1 or seb1
seb2
and the
exocyst mutants (sec3-101, sec5-24, sec6-4, sec8-9, sec10-2, and
sec15-1). Deletion of either SEB1 or its close homologue
SEB2 does not create a phenotype, but simultaneous deletion of both
genes results in temperature sensitivity at 38 °C
(20,
22). All of the exocyst
mutants were crossed with seb1
or with seb1
seb2
strains followed by tetrad analysis. No interactions were
observed between seb1
and exocyst mutants. The triple mutants
seb1
seb2
sec10-2 and
seb1
seb2
sec15-1 derived from
crosses of the double deletant seb1
seb2
together with sec10-2 or with sec15-1 mutation were 1-2
°C more temperature-sensitive than the seb1
seb2
double mutant or the single mutants sec10-2 and
sec15-1 (data not shown). The suppression data and the synthetic
interactions demonstrate multiple genetic interactions between the Sec61
and the exocyst complex.
Exocytosis Mutations Are Suppressed by Overexpression of the Sec61p Complex Components but Not Vice VersaAs multiple genetic interactions were observed for SEB1 and the exocytosis machinery, we studied whether overexpression of the other ER translocation components could suppress mutations in the late-acting SEC genes. The exocyst mutant strains were transformed with plasmids harboring SEC61, SSS1, SEC62, SEC63, or SEB2 or with their vector controls, and the growth of the transformants was tested at different temperatures. SEC61 rescued the growth of sec3-2 efficiently and the growth of sec5-24 and sec6-4 partially (Fig. 2A). Overexpression of SSS1 rescued the growth of sec8-9 and sec15-1 efficiently and growth of sec3-2 and sec5-24 partially (Fig. 2B). In contrast, overexpression of SEC62, SEC63, or SEB2 did not suppress the ts defects in any of the exocytosis mutants tested (data not shown).
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Suppression of the other late-acting secretory mutants (sec1-1,
sec2-41, sec4-8, sec9-4, and sso2-1), mutants of the general
components (sec17-1, sec18-1, and sec19-1), as well as
mutants of SEC1 homologues (sly1-1 and slp1)
were also analyzed in this screen. Weak suppression by SEB1
overexpression was observed in sec2-41 and in sec19-1
mutants (data not shown). No interactions were detected with any of the other
genes studied. The effect of SEB1 overexpression in kar2-133,
kar2-159, and sec13-1 mutants was also tested, but their growth
at the restrictive temperature was not rescued. Thus, the genetic interactions
observed were most prominent between the genes encoding components of the two
multiprotein complexes, the Sec61p translocation complex and the exocyst.
To test whether the observed genetic interactions function also in the opposite direction, the possible suppression of defects in the ER translocation components by overexpression of genes encoding components of the exocytosis machinery was studied. The sec61-1 and sec61-2 mutants are temperature-sensitive at 37 °C (46, 47), and the sec61-32 and sec61-41 mutants are cold-sensitive at 17 °C (48). Their growth at these temperatures was not rescued by overexpression of SEC1, SEC3, SEC15, SSO1, or SSO2 gene (data not shown). The double deletion of both SEB1 and SEB2 results in impaired growth at 38 °C (20, 22). The growth of this double mutant was not rescued by overexpression of any of the late-acting secretion genes tested. Thus, the suppression is unidirectional; only defects in the exocytosis machinery are corrected by increased amounts of the ER translocon components and not vice versa.
The Seb1p Transmembrane Domain Is Required for sec15-1
SuppressionSeb1p is a membrane protein with a single C-terminal
transmembrane domain (20). To
test whether the suppression activity of Seb1p requires the transmembrane
domain and thus tight membrane association, a mutant version of Seb1p lacking
the hydrophobic C-terminal membrane anchor was created. The sec15-1
cells were transformed with the plasmid encoding the mutant, YEpSEB1-MA, the
plasmid encoding the wild type SEB1, or the empty vector, and the
growth of the transformants was monitored at different temperatures. The
Seb1p-MA did not suppress the temperature sensitivity of sec15-1 even
at 35 °C, the lowest temperature restrictive for sec15-1 cells
(Fig. 3A). The
Seb1p-MA mutant did not affect the cell growth at the permissive temperature
(24 °C), indicating that its expression is not harmful
(Fig. 3A). The lack of
a phenotype for the Seb1p-MA overexpression could be because of instability of
the mutant protein. To test this possibility, lysates were prepared from the
transformants used in the suppression experiment, and the amounts of Seb1p and
Seb1p-MA were studied by Western blotting with Seb1p-specific antibodies.
Quantitation of the blots showed that the wild type Seb1p was 14 times
and the Seb1p-MA 10 times overexpressed in comparison to the endogenous Seb1p
level (Fig. 3B). This
indicates that Seb1p-MA is stably expressed in sec15-1 cells but
fails to multicopy suppress this mutant.
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Seb1p Coimmunoprecipitates with the Exocyst Components Sec8p and Sec15p in Yeast Cell LysatesThe genetic interactions between SEB1 and the exocytosis machinery prompted us to study protein-protein interactions between Seb1p and the exocyst proteins. For detection of Sec8p, a yeast strain, H2647, was used in which the only chromosomal copy of SEC8 gene encodes a protein tagged with three Myc epitopes at its C terminus (1). This strain was transformed either with a multicopy plasmid expressing HA-tagged SEB1 from the ADH1 promoter (20) or with an empty vector. In parallel, wild type strain NY15 was transformed with the same plasmids. The HA-tagged form of Seb1p is fully functional such that its overexpression suppresses the ts growth defects of sec15-1, sec10-2, and sec8-9 like the wild type gene (data not shown) and was utilized in these experiments to detect the overexpressed Seb1p. Antibodies against the N-terminal peptide of Seb1p coprecipitated the Myc-tagged Sec8p (Fig. 4, top panel). The Seb1p antibodies precipitated 1.6% of the Sec8-Myc protein present in the cell lysate used for precipitation. The coprecipitation of Sec8p-Myc was genuinely due to the presence of the Seb1p antibodies as evidenced by the absence of Sec8p-Myc in the mock precipitations carried out in the absence of the Seb1p antibodies (data not shown) or in the presence of the preimmune serum (Fig. 4, top panel, pis IP). In the Western blots the anti-Myc antibodies also detected a slightly faster migrating protein band, which may be a proteolytic cleavage product of the tagged Sec8p. The Seb1p antiserum immunoprecipitated both the HA-tagged and the wild type Seb1p (Fig. 4). The antibodies against Sec15p coprecipitated the endogenous Seb1p rather inefficiently (Fig. 4, middle panel). Sec15p antibodies brought down 0.6% of the Seb1p present in the input of the immunoprecipitation. More of the HA-tagged form of Seb1p was detected, as it was overexpressed in these transformants. However, the endogenous Seb1p also was precipitated more efficiently in the overexpressing cells, suggesting increased stability of the complex containing Seb1p and Sec15p (Fig. 4, middle panel). In the Sec15p immunoprecipitations, the Myc-tagged Sec8p was precipitated from lysates of H2647 transformants as expected because both of these proteins are present in the exocyst complex.
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Coimmunoprecipitation of Seb1p and Sec4pSec15p binds the small GTPase Sec4p (49). To study whether Sec4p is present in the complex containing Seb1p, Sec8p, and Sec15p, we performed immunoprecipitations with Sec4p-specific antibodies. H2647 or wild type cells transformed with a plasmid overexpressing HA-SEB1 or with the empty vector were lysed and subjected to immunoprecipitations with antibodies against Sec4p. Seb1p coimmunoprecipitated with Sec4p as did the Sec8p-Myc, indicating that the complex that is formed by the exocyst components and Seb1p also contains Sec4p (Fig. 4, bottom panel). The preimmune serum did not precipitate Seb1p, Sec8p-Myc, or Sec4p.
To rule out the possibility that Seb1 protein is brought down nonspecifically with immunocomplexes formed in the yeast cell lysates, two cell surface proteins not related to the exocytosis machinery were used as controls. Thus, immunoprecipitations with antibodies against Pma1p, an abundant plasma membrane protein (50), and Gas1p, a glycosylphosphatidylinositol-anchored plasma membrane/cell wall protein (51), were performed. Neither one was able to coprecipitate Seb1p (data not shown), which further indicates that Seb1p immunoprecipitation together with the exocyst components and Sec4p is not because of nonspecific cross reactions.
Overexpression of SEB1 Recovers Seb1p-Sec15p Interaction in sec15-1 CellsAs SEB1 was isolated originally as a multicopy suppressor able to rescue the growth of sec15-1 cells at the restrictive temperature, we next tested whether the putative Seb1p-Sec15p complex is present in the sec15-1 mutant cells. The sec15-1 cells overexpressing SEB1 or those carrying the empty vector plasmid were grown to early logarithmic growth phase at 24 °C, and the cultures were divided in half. One-half was shifted to 38 °C for 30 min before cell lysis, while the other remained at 24 °C. Immunoprecipitations were performed with polyclonal Sec15p antibodies. Interestingly, Seb1p could not be coprecipitated with Sec15p antibodies from the cells harboring the control plasmid even at the permissive temperature (24 °C) (Fig. 5). From transformants overexpressing SEB1, efficient precipitation of Seb1p was obtained with the Sec15p antibodies. More importantly this precipitation was obtained also from cells that had been incubated at the restrictive temperature, 38 °C. The Sec15p preimmune serum did not precipitate Seb1p from any of the transformants (Fig. 5). These results suggest that the Sec15-1 mutant protein is partially defective even at the permissive temperature, that an increased level of Seb1 protein restores the formation of the putative Seb1p-Sec15p complex with the mutant protein, and that this happens also at the restrictive temperature.
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Overexpression of SEB1 or SEC4 Increases Production of Secreted Proteins in Wild Type YeastThe above genetic interaction results suggest that SEB1 overexpression facilitates exocytosis. We have shown previously that overexpression of the SSO1 and SSO2 genes encoding plasma membrane t-SNAREs (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors), essential for exocytosis, increases the production of secreted proteins (42). We therefore proceeded in studying the effect of SEB1 overexpression on production of secreted proteins in wild type cells. Secretion of the yeast endogenous enzyme invertase was studied in DBY746 cells grown in 2% sucrose medium to allow expression of the glucose repressed SUC2 gene. Secretion of invertase to the cell surface was analyzed at several time points during growth in liquid medium, and reproducibly, up to 40% higher invertase activity was detected with SEB1 overexpression. This is the same level that is obtained with SSO2 overexpression from the endogenous SUC2 gene (42).
We also studied the effect of SEB1 overexpression on secretion of
a heterologous reporter protein, Bacillus -amylase
(42). The
-amylase gene
was integrated into the yeast genome at the TRP1 locus. This strain,
H1256, was transformed with YEpHA-SEB1 or the empty vector, pVT102U. The
transformants were grown in shake flasks in selective medium, and the amount
of
-amylase secreted into the culture medium was determined. In
comparison with the control transformant, up to 3-fold more
-amylase
activity was found in the medium of SEB1 overexpression strain
depending on the cultivation time point analyzed
(Fig. 6A). The
increased
-amylase activity in the medium was due to increased amount
of the enzyme as determined by Western blotting (data not shown). To verify
whether the increased production of
-amylase was due to increased
transcription of the gene, the
-amylase mRNA levels were determined by
Northern blotting. The
-amylase transcript level was increased slightly
in the SEB1 overexpressing strain, being about 1.5-fold compared with
that in the control strain. We next studied the effect of overexpression of
SEC4 on
-amylase production. Sec4p is a small GTPase that is
essential for regulation of exocytosis and binds to the post-Golgi secretory
vesicles and Sec15p. Similar to SEB1, overexpression of SEC4
resulted in 3-fold increase in production of secreted
-amylase
(Fig. 6B).
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-Amylase is secreted efficiently by the wild type yeast. About 75%
of the enzyme activity is found in the culture medium and in the periplasmic
fraction/cell wall (42).
Fractionation of the yeast cells to the periplasmic and cytoplasmic fractions
was performed for SEB1-overexpressing and control cells to quantitate
the total amount of exocytosed
-amylase. The enhancement of the
production of secreted
-amylase was 2.3-fold
(Table II). The overall
-amylase level in the SEB1 transformant was about 2-fold
higher, and 90% of the enzyme was secreted instead of 75% of the enzyme
present in the control strain.
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DISCUSSION |
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Here we show that overexpression of SEB1 suppresses the ts
mutations in all SEC genes encoding components of the exocyst
complex. A functional link between the ER translocon and the exocyst complex
is further supported by the increased temperature sensitivity detected in
seb1 seb2
sec10-2 and in
seb1
seb2
sec15-1 triple mutants.
Although this synthetic effect was not strong, it may be important, because
Sec10p and Sec15p form a subcomplex in vivo suggested to bridge the
transport vesicle with the remaining exocyst components
(49). Overexpression of the
other two genes encoding components of the Sec61p complex, SEC61 and
SSS1, also suppressed the ts defects in several of the exocyst
mutants. The other late-acting mutations suppressed by SEB1
overexpression were sec2-41 and sec19-1 (data not shown).
The Sec2p and Sec19p are regulators of the Sec4p, a Rab GTPase that interacts
physically with Sec15p (49).
These genetic interactions strongly suggest closer functional connection
between the ER translocation complex and the exocytosis machinery than was
surmised before.
The role of the nonessential subunits of the Sec61p and Ssh1p
complexes in translocation is not known. Disruption of both SEB1 and
SEB2 results in only a slight reduction in protein translocation and
Gas1p maturation
(22).2
The mammalian Sec61
is not essential for translocation but has been
shown to facilitate this process
(52). In yeast, overexpression
of SEB1 suppresses the sec61 mutations that destabilize the
Sec61p structure (20,
33,
53). However, mutations that
do not affect the stability of Sec61p, but render it nonfunctional in
translocation, are not suppressed by SEB1
(54). This suggests an
accessory or stabilizing role for the
subunit in the complex.
The overexpression suppressions reported here show strict directionality; only the ER translocon components can rescue the function of the mutated exocyst components and not vice versa. Thus, the observed genetic interactions and the rescued physical interaction of Seb1p with Sec15-1 mutant protein in SEB1 overexpressing cells (Fig. 5) suggest that increased Seb1p level facilitates directly or indirectly the function of the destabilized (2, 55, 56) exocyst complex in the mutant cells. Coprecipitation of Seb1p with exocyst components Sec8p and Sec15p, and the Sec15p-interacting protein Sec4p, implies that these proteins can be present in the same protein complex within the yeast cell. Notably, these coprecipitations were detected also in cells expressing the endogenous level of Seb1 protein. Under the experimental conditions used here the proportion of Seb1p coimmunoprecipitating with Sec8p or Sec15p was low. The interactions were, however, specific as shown by the control experiments performed. The low coimmunoprecipitation efficiencies may be due in part to the fact that Seb1p may not need to exist in a stable complex with exocyst components to fulfill its biological function. We favor the view that the observed interaction reflects the existence of a regulatory signal between the two endpoints of the secretory pathway. Such interactions are likely to be transient by nature and do not necessarily require simultaneous association of a large proportion of the cellular Seb1p with exocyst subcomponents. We would also like to stress that the fact that Seb1p overexpression rescued the interaction with the mutant Sec15p and the cell growth indicates a direct or indirect functional interaction between these molecules. Additional support for the interaction is provided by the extensive genetic data and the observed secretion enhancement, which collectively point to an in vivo interaction of these processes.
The interactions between the ER translocon and the exocyst components are intriguing because functionally the Sec61p translocation complex and the exocyst are located at opposite ends of the secretory pathway. It has, however, been shown that the cortical ER membrane and the plasma membrane frequently run in parallel in yeast cells (57, 58). Thus, at least a portion of the Sec61p and the exocyst complexes can be in close proximity with each other within a cell. Lipschutz et al. (Ref. 65; accompanying article) demonstrate that ER translocation machinery components can exist in a complex with components of the exocyst also in Madin-Darby canine kidney cells. Interactions between the exocyst and ER resident proteins are supported also by the observed coimmunoprecipitation and partial colocalization of the mammalian Sec8p with the inositol 1,4,5,-trisphosphate receptor, IP3R (59).
The physical interactions with the exocyst complex both in yeast and
mammalian cells were studied only with the subunit of the ER
translocon. In these experiments several components (Sec6, Sec8, Sec10, and
Sec15) of the exocyst complex were detected. Although our genetic data suggest
that all components of these complexes may have roles in these interactions,
the
subunit clearly seems to play the most important role. Increased
production of secreted proteins was achieved by overexpression of
SEB1 only, not with that of SEC61 or
SSS1,3 and
SEB1 was most efficient also in the suppression of the exocyst
mutations. Overexpression of a mutant form of Seb1p lacking the transmembrane
domain failed to suppress sec15-1 temperature-sensitive growth. This
suggests that association of Seb1p in the ER membrane may be required to
establish the functional link with the exocyst complex. Presently, we do not
know the proportion of Seb1p in the translocating Sec61p complexes in
SEB1 overexpressing cells. As the amount of Sec61p or Sss1p does not
increase in an Seb1p-overexpressing strain (data not shown), a portion of
Seb1p may exist outside of these complexes under these conditions. The
possible contribution of the unassembled Seb1p to the interaction remains to
be studied. It may be that Seb1p interaction with the exocyst is not direct
but is mediated by other, presently unknown factors.
In addition to the observed physical and genetic interactions, an
additional functional link was observed between the early and late processes
of protein secretion. Both overexpression of Seb1p and the Sec15p-interacting
protein, Sec4p, resulted in enhanced protein secretion in yeast. Increased
production of secreted proteins in yeast can occur as a result of ER
proliferation caused by overexpression of a heterologous ER membrane protein
(60,
61). On the basis of previous
morphological experiments, even the strongly overexpressed Seb1p is localized
in the ER (20). When analyzed
by electron microscopy, SEB1 overexpression does not cause ER
membrane proliferation nor does overexpression of Seb1p increase the levels of
Sec61p or Sss1p (data not shown). Lipschutz and colleagues
(62) have shown that
hsec10 overexpression in Madin-Darby canine kidney cells increases
the amount of secreted and basolateral membrane proteins. Similarly, we show
here that overexpression of Sec4p, a Sec15p-interacting protein, results in
enhancement of protein secretion. The overexpression of SEC15 is
toxic to yeast cells (39).
Thus its possible enhancing effect on secretion could not be studied. In an
accompanying article, Lipschutz et al.
(65) now show an unaltered
transcript level of proteins with increased secretion levels. Likewise,
enhanced production of -amylase in SSO1 or
SSO2-overexpressing cells is not because of increased transcription
of the
-amylase gene
(42). Taken together, these
results suggest that the increased production of secreted proteins in the
overexpression strains occurs at post-transcriptional level.
In mammalian cells the Sec61 subunit has been shown to bind
nontranslating ribosomes (63).
Thus, it is possible that increased amount of the
subunit facilitates
ribosome binding to ER and thereby increases the number of ribosomes that are
available for binding and translation of the messengers of secreted proteins
in SEB1 overexpressing cells. This perhaps explains the observed
small increase in the
-amylase transcript level in SEB1
overexpressing yeast cells. Efficient ribosome binding and ER targeting have
been shown to increase the half-life of mRNAs in yeast
(63,
64). Although a link between
the ER translocation machinery and exocyst is observed both in yeast and in
mammalian cells, differences in forming and maintaining this link are likely
to exist because of the different cellular signals that these cells encounter
in their growth environments.
The functional and physical interactions between the ER translocon and the exocyst complex indicate the existence of a regulatory circuit that may be used for adjusting the level of translation to the secretory capacity of the cell. Increasing the level of a component involved in this regulation could lead to enhanced production of secreted proteins in wild type cells. Along the same lines, this cross-talk would be compromised by a decreased amount of (or defective) regulatory component, e.g. destabilized exocyst complex in the mutant cells. Increasing the amount of an interacting component, in this case Seb1p, could restore sufficient communication even in mutant cells and allow growth at the nonpermissive temperature. This view is supported by the fact that Sec15-1p and Seb1p were found in the same protein complex only when SEB1 was overexpressed (Fig. 5).
Taken together, our data reported here reveal novel links between molecular complexes of the ER translocon and the exocyst complex, implying new regulatory circuits between the ER and plasma membrane in the control of protein secretion in yeast.
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FOOTNOTES |
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To whom correspondence may also be addressed. Tel.: 358-9-4565225;
Fax: 358-9-4552103; E-mail:
jussi.jantti{at}vtt.fi.
To
whom correspondence may be addressed. Tel.: 358-9-4565138; Fax: 358-9-4552103;
E-mail:sirkka.keranen{at}vtt.fi.
1 The abbreviations used are: ER, endoplasmic reticulum; ts, temperature
sensitive; HA, hemagglutinin; Pipes, 1,4-piperazinediethanesulfonic acid.
2 J. Toikkanen and S. Keränen, unpublished observation.
3 J. Toikkanen, H. Söderlund, and S. Keränen, manuscript in
preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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