(Received for publication, July 26, 1995; and in revised form, February 6, 1996)
From the
Signal recognition particle-dependent targeting of secretory
proteins to the endoplasmic reticulum membrane is predominant in the
yeast Yarrowia lipolytica. A conditional lethal mutant of the SCR2-encoded 7S RNA provided the first in vivo evidence for involvement of this particle in cotranslational
translocation (He, F., Beckerich, J. M., and Gaillardin, C. M.(1992) J. Biol. Chem. 267, 1932-1937). In order to identify
partners of 7S RNA or signal recognition particle in their function, we
selected synthetic lethal mutations with the 7S RNA mutation (sls). The SLS1 gene, cloned by complementation of
the sls1 mutant growth defect, encodes a 426-amino acid
polypeptide containing a NH-terminal signal peptide and a
COOH-terminal endoplasmic reticulum (ER) retention motif. The SLS1 gene product behaves as a lumenal protein of the ER. Sls1p was
sedimented with membrane-rich organelles and was resistant to protease
degradation without prior membrane solubilization. Immunofluorescence
microscopy showed a typical endoplasmic reticulum perinuclear staining.
Co-immunoprecipitation revealed that Sls1p resides close to the major
translocation apparatus component, Sec61p. Deletion of the SLS1 gene led to a temperature-sensitive growth phenotype. Synthesis of
several secretory proteins was shown to be specifically reduced in
sls1 cells. We propose that Sls1p acts in the preprotein
translocation process, interacting directly with translocating
polypeptides to facilitate their transfer and/or help their folding in
the ER.
In order to enter the secretion pathway, secretory proteins of
eukaryotic cells have to be transported across or inserted into the
endoplasmic reticulum (ER) ()membrane. To achieve this
translocation step, secretory proteins must be specifically targeted to
the translocation machinery in the ER membrane and be competent for
crossing this membrane(2) . In higher eukaryotes, the signal
recognition particle (SRP) was shown to take part into these
functions(3) . SRP is composed of a single 7S RNA and six
polypeptides (4) . When the signal sequence of a nascent
secretory polypeptide is extruded from the ribosome, it is first
recognized by the nascent polypeptide associating complex(5) ,
which allows specific binding of SRP. Interaction of SRP with the
nascent chain-ribosome complex causes translational slow down. After
binding of SRP to its membrane-bound receptor, SRP is displaced from
the complex and the nascent chain is transferred to the translocation
site where crossing takes place simultaneously to translation. As soon
as the polypeptide emerges in the lumen of the ER, it interacts with
various proteins for processing and folding. A somewhat different
picture emerged from studies on the yeast Saccharomyces
cerevisiae. Indeed, several secretory proteins in this yeast
appeared to be transported post-translationally, both in vivo and in vitro(6, 7) , and homologues of
mammalian SRP components that have been identified in this yeast and
function in translocation (8, 9, 10, 11) are not essential for
cell viability. In another yeast Yarrowia lipolytica, deletion
of both genes SCR1 and SCR2 encoding 7S RNA is
lethal(12) , and we suggested earlier that the SRP-dependent
targeting may be the main pathway, as in higher eukaryotic cells.
Isolation of conditional lethal mutants in the 7S RNA provided in vivo evidence for involvement of SRP in cotranslational translocation(1, 13) . In order to identify partners of SRP in this process and to better understand its molecular mechanisms, we have now selected synthetic lethal mutations with the 7S RNA mutation, called sls. In the present paper, we describe identification and characterization of one of these genes, SLS1, and of its gene product.
The Y. lipolytica haploid mutagenized strain was MatB, scr1::ADE1, scr2, ura3, leu2, his-1, containing the replicative plasmid pINA1090 carrying
the scr2-II.13 allele and the URA3 gene. Replacement
of the scr2-II.13 allele by the wild-type allele was done by
plasmid shuffling using the replicative plasmid pINA237, which
contained the SCR2 gene and the LEU2 gene. For
genetic studies, the sls1 Ts mutant was mated with a MatA, scr1::ADE1,
scr2, lys11, ura3, leu2 strain containing the plasmid pINA398
carrying the SCR2 gene and the URA3 gene. Diploids
were sporulated and analyzed as described previously(12) . To
isolate the SLS1 gene from a LEU2-based replicative
genomic library constructed by P. Fournier, a Leu- Ts segregant
from this cross was retained. Transformation of Y. lipolytica by the lithium acetate method was performed as described
previously(14) . Y. lipolytica strains were usually
grown at 28 °C in YPD (1% yeast extract, 1% bacto-peptone, 1%
glucose). For transformant selection, minimal medium (0.17% yeast
nitrogen base without ammonium sulfate and without amino acids, 1%
glucose, 0.1% proline) was used, and supplements were added to a final
concentration of 0.01%. 5-Fluoroorotic medium contained 0.001% of
uracil and 0.125% of 5-fluoroorotic acid. AEP induction was performed
using GPP medium (2% glycerol, 0.17% yeast nitrogen base without
ammonium sulfate and without amino acids, 0.3% proteose peptone, 50
mM phosphate buffer, pH 6.8, and appropriate supplements for
the growth of cells). For labeling, GC medium (same as GPP but 0.2%
casein instead of 0.3% proteose peptone) was used.
Figure 1: Colonies formed on rich medium after incubation at 28 °C by the double mutant sls1-1, src2-II.13 (1), the sls1-1 mutant (2), and the wild-type strain (3).
Figure 2: Map of the chromosomal insert cloned in the plasmid complementing the sls1 mutant temperature sensitivity (1) and subfragments tested for complementation of the sls1-1 growth defect in multicopy (2-5) or monocopy (6). C, ClaI; E, EcoRI; H, HindIII; N, NheI; P, PvuII; S, SalI; Ss, SspI; Xb, XbaI.
Figure 3: A, detailed restriction map of the sequenced minimal complementing region. The SLS1 open reading frame is represented by a large dark arrow. The upstream and downstream regions are visualized by a thin box. The ClaI-HindIII fragment containing a internal deletion and the URA3 gene was used to inactivate the chromosomic copy of SLS1. B, nucleotide sequence of the SLS1 gene. Coordinate +1 corresponds to the translation initiation codon. The potential TATA box is circled, and the potential polyadenylation site is underlined. The predicted amino acid sequence is shown in single-letter code. The putative signal peptide and ER retention motif are boxed.
Figure 4:
Localization of the Sls1 protein. A, characterization of the anti-Sls1p antibodies produced
against a glutathione S-transferase-Sls1p fusion protein.
Western blot analysis of whole cell extracts from various Y.
lipolytica transformants containing different SLS1 mutant
constructs. The strain in lane 1 contains the wild-type SLS1 allele on the chromosome, the strain in lane 2 also contains the SLS1 gene on a 3-4-copy plasmid,
the strain in lane 3 contains the deleted sls1 allele on the chromosome, and the strain in lane 4 contains a 3`-truncated chromosomal copy of SLS1. Equal
amounts of total protein were applied on SDS-PAGE. B, cell
fractionation and protease protection of Sls1p. A whole cell extract
from the wild-type strain 136463 was made by a gentle method and
cleared from unbroken cells and cell wall fragments by centrifugation
at 450
g. Supernatant (S
) was
recollected and subfractionated by a 20-min centrifugation at 10,000
g leading to two fractions (S
and C
). Samples of the resuspended
latter fraction were treated on ice for 1 h with 0.5 mg/ml of
proteinase K in the absence or in the presence of 4% Triton X-100.
Samples were analyzed by SDS-PAGE followed by Western blotting with
Sls1p antiserum. C, localization of Sls1p by
immunofluorescence in cells expressing the chromosomic SLS1 gene. Cells were grown overnight in rich medium, prepared for
immunofluorescence, and treated with rabbit anti-Sls1p antibodies,
followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG to
localize the Sls1 protein (a) and 4,6-diamidino-2-phenylindole
to visualize nuclear DNA (b and d). The Sls1p signal
was abolished by exclusion of the primary antibodies or by staining of
the
sls1 strain (c).
Figure 5: Levels of intracellular Sls1p after heat shock and tunicamycin treatment. A, half of an overnight culture in rich medium was incubated at 34 °C (lanes 2, 4, and 6), whereas the second half remained at 28 °C (lanes 1, 3, and 5). Samples were taken every 15 min for 90 min. B, half of an overnight culture in rich medium was incubated for 3 h at 23 °C in the presence of tunicamycin (10 µg/ml) (lane 2), whereas the second half was left untreated (lane 1). Whole cell extracts were made and subjected to SDS-PAGE (8% polyacrylamide gel). Proteins were transferred to mitrocellulose and probed with rabbit anti-Sls1p antibodies and goat anti-rabbit IgG conjugated with peroxidase. In these experiments, equal amounts of total protein were loaded for each extract.
sls1 and SLS1 cells were labeled in inducing medium for 45 s
and chased. Fig. 6A shows a 7-fold reduction of the
amount of labeled intracellular forms in
sls1 cells
compared with SLS1 cells (lanes 1-4 versus lanes
5-8). The level of the secreted 32-kDa mature form in the
growth medium of
sls1 cells, detected after
trichloroacetic acid precipitation, was also lower than these revealed
in supernatant of SLS1 cells (Fig. 6B, lanes 1-4 versus lanes 5-8). Total protein
synthesis was similar in the two strains. Because AEP precursors in
sls1 cells could bind to concanavalin A-Sepharose (not
shown), they corresponded to translocated forms. Maximal level of both
precursor and mature forms was only obtained at 2.30 min in
sls1 cells (Fig. 6A, lane 3)
compared with 1 min in SLS1 cells (Fig. 6A, lane 6). Therefore, detection of total newly synthesized AEP
precursors was delayed in the absence of the SLS1 gene
product. This observation suggests that translocation is affected in a
sls1 context. In addition, a delay in AEP processing was
observed. In contrast to SLS1 cells, where the mature form was
predominant at 1 min in cell extracts (Fig. 6A, lane 6) and secreted at 2.30 min in the medium (Fig. 6B, lane 7), most of AEP mature form was
immunoprecipitated at 2.30 min in
sls1 cell extracts (Fig. 6A, lane 3) and appeared at 5 min in
supernatant (Fig. 6B, lane 4). This delay in
maturation reflects an increased transit time upstream from the Golgi.
Figure 6:
Pulse-chase labeling and
immunoprecipitation analysis of AEP synthesis and secretion in SLS1 and sls1 strains. Y. lipolytica SLS1 cells (lanes 5-8) and
sls1 cells (lanes
1-4) were grown overnight at 18 °C in inducible medium,
concentrated in fresh medium, and transferred for 1 h at 28 °C
before a 45-s labeling pulse with L-[4,5-
H]leucine. Cells were chased by
the addition of a 300-fold excess of cold leucine, and samples were
taken 0, 1, 2.30, and 5 min after the chase. A, intracellular
extracts were immunoprecipitated with polyclonal anti-AEP antibodies
and protein A-Sepharose. B, supernatant proteins were
trichloroacetic acid-precipitated. Precipitates were resolved by
SDS-PAGE and visualized after fluorography at -80 °C.
Positions of the AEP 55-kDa precursor and the 32-kDa mature form are
indicated by arrows.
The effect of the deletion of the SLS1 gene on other
secretory proteins was tested by looking at the amount of glycosylated
[S]methionine-labeled proteins bound to
concanavalin A-Sepharose in
sls1 cells compared with SLS1 cells. The level of several major glycoproteins appeared
to be largely reduced in the absence of the SLS1 gene product (Fig. 7, lane 2 compared with lane 1), suggesting that
synthesis of these preproteins was impaired. In contrast, the amount of
at least one glycoprotein was increased in
sls1 cells.
Figure 7:
Pattern of glycosylated proteins in SLS1 cells (lane 1) and in sls1 cells (lane 2) at 26 °C.
[
S]Methionine-labeled total proteins from each
strain were incubated with concanavalin A-Sepharose beads. Bound
proteins were eluted by heating, applied to a 6% SDS-polyacrylamide
gel, and visualized after fluorography.
Figure 8: AEP secretion in Sls1p overexpressing cells. SLS1 monocopy cells (lanes 1-4) and SLS1 multicopy cells (lanes 5-8) were pulse-labeled for 45 s and chased as previously described. Samples were taken at 0, 1, 2.30, and 5 min post chase and centrifuged. Proteins from each supernatant were trichloroacetic acid-precipitated and subjected to SDS-PAGE and fluorography. Mature AEP is signaled by an arrow.
Figure 9:
Sls1p and Sec61p co-precipitation. A, a membrane-rich fraction was prepared from wild-type cells.
Samples were subjected or not to cross-linking by the cleavable reagent
dithiobis(succinimidyl propionate) (0.2 mg/ml) simultaneously to
solubilization in 1% Triton X-100 and were immunoprecipitated by
anti-S. cerevisiae Sec61p antibodies. Precipitates were
resolved on SDS-PAGE, transferred to nitrocellulose, and blotted with
anti-Sls1p antibodies. Lane 1, crude extracts; lane
2, Sec61p immunoprecipitates after cross-linking; lane 3,
Sec61p immunoprecipitates without dithiobis(succinimidyl propionate)
treatment. B, Sec61p blotting with antibodies raised against a
21-amino acid NH-terminal peptide
on crude
extracts (lane 1), on extracts solubilized with 1% Triton
X-100 and immunoprecipitated either by anti-Y. lipolytica Sec61p antibodies (lane 2), by anti-S. cerevisiae Sec61p antibodies (lane 3), or by anti-Sls1p antibodies (lane 4). In lane 5, immunoprecipitates analyzed in lane 2 were probed with anti-Y. lipolytica Sec61p
preimmune serum.
Genetic data indicate that major secretory proteins are targeted and translocated across the ER membrane in a SRP-dependent way in the yeast Y. lipolytica(12) . This yeast therefore represents a good model for a genetic approach of cotranslational translocation molecular mechanisms. A SRP-deficient strain carrying the mutant scr2-II.13 allele on a replicative plasmid (1) was used to look for secondary mutations that specifically exacerbate the Ts growth phenotype displayed by this first mutation. Such genetic interactions leading to synthetic enhancement have been observed a posteriori for SEC17 and SEC18, whose products are both involved in transport vesicles fusion(17) , or between two specific alleles of the SEC63 and KAR2 genes(26) . In our case, secondary mutations could affect products that either act at the same targeting step or function in a different but coordinated step. Several mutations were obtained that conferred high sensitivity to elevated temperature only in combination with the 7S RNA mutation (sls); one called sls1-1 was studied in more detail.
The SLS1 gene
encoded a 426-amino acid residue polypeptide with a functional signal
sequence at its NH terminus and a functional ER retention
signal at its COOH terminus as evidenced by immunofluorescence
microscopy. Like lumenal ER proteins in other yeasts, the Y.
lipolytica Sls1p hydrophilic protein is probably localized into
the ER through a retention mechanism involving a specific
receptor(27) . This receptor should recognize the RDEL motif
and allow retrieval of the protein from a late compartment. Indeed,
removal of this ER retention signal results in decreased amounts of
intracellular Sls1p. The truncated protein probably continues on the
secretory pathway transiting through the Golgi and secretion vesicles.
However, no significant levels of Sls1p were detected in the growth
medium, probably due to instability of the protein outside in the
medium or degradation along the secretory pathway. The strain
expressing this truncated Sls1 protein did not show any growth defect,
suggesting that the secreted form of Sls1p performed its function
during its transit through the ER.
The sls1 null mutation
conferred a temperature-sensitive growth phenotype similar to that of
the sls1-1 mutant, indicating that the 20-kDa SLS1 product detected in the latter cells was not functional. The
temperature sensitivity of sls1 cells may reflect an
improving role for Sls1p in an essential cellular process, such as
preprotein translocation, that could become limiting at 30 °C in
the absence of Sls1p. Alternatively, Sls1p may perform an essential
function only at elevated temperatures in a heat shock pathway. In a
sls1 context, synthesis of several preproteins was
severely reduced as in the scr2 mutant. The main conclusion
from this observation is that the Sls1 protein is likely to be involved
in the cotranslational translocation process, accounting for the
genetic interaction between the two mutations. The translation defect
of the scr2-II.13 mutant could be explained by a decrease in
initiation of preprotein translocation. To explain the translation
defect in the absence of Sls1p, we favor the hypothesis that Sls1p
helps translocating polypeptides on the lumenal side of the ER
membrane, completing their translocation. Consistent with this scheme,
a delay is observed for the appearance of total newly synthesized
precursors in the absence of the SLS1 gene product. The
function of mammalian ER lumenal proteins in cotranslational
translocation, mediating the net transfer of the polypeptide into the
ER lumen, has already been proposed(28) . Sls1p might thus
increase the translocation initiation rate on the opposite side of the
ER membrane by freeing the translocon from the ER side.
Co-precipitation of Sls1p with the major component of the translocation
apparatus, which reveals that Sls1p resides in the vicinity of the
translocation site, fits well with a function of the protein in
preprotein translocation. Interestingly, Sls1p displays a domain
similar to the COOH-terminal moiety of the family of Sec61
polypeptides (Fig. 10). Sec61
was described as a companion
of Sec61p (Sec61
) in the translocon(29) . We propose that
this domain is involved in Sec61p binding for Sls1p. This hypothesis
will be tested in the near future.
Figure 10:
Sequence alignment of a Sls1p domain with
a region of Sec61 proteins from various organisms and human cDNA
sequences. Identical amino acids are shaded in dark gray and similar residues in light
gray.
Our results are compatible with a
second function of Sls1p in protein folding after translocation has
been completed, its defect accounting for the delay observed in the
processing of the 55-kDa precursor form in sls1 cells.
Additional evidence for a role of Sls1p during precursor transit
through the ER was provided by the delay of precursor processing into
the mature form in cells overexpressing Sls1p. Prevention of secretion
of some secretory proteins has been described in mammalian cells
overexpressing the BiP protein (30) and was supposed to result
from the stabilization of the complex between newly translocated
preprotein and BiP. Induction of Sls1p levels under conditions leading
to ER accumulation of misfolded preproteins was also consistent with
such a property.
Our results show that synthetic lethality may be used as a screen for isolation of new mutations and that such genetic interaction could be observed for two products that are physically separated but involved in the same coordinated pathway.