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J Biol Chem, Vol. 273, Issue 47, 30903-30908, November 20, 1998
,From the Laboratoire de Génétique Moléculaire et Cellulaire, INRA, CNRS, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France and the § Georg-August Universitaet Goettingen, Zentrum Biochemie und Molekular Zellbiologie, 37073 Goettingen, Germany
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The yeast Yarrowia lipolytica is a
model organism for in vivo study of the signal recognition
particle-dependent targeting pathway. In this report, we
defined solubilization conditions and set up a fractionation procedure
of Y. lipolytica microsomes to determine the amounts of
Sec61p-containing translocation pores linked to ribosomes. In contrast
to Saccharomyces cerevisiae, from 70 to 80% of Sec61p
associates with ribosomes in this yeast. The chaperone protein Kar2p
and the Sls1p product, a resident protein of the endoplasmic reticulum
lumen, partially fractionate with this Sec61p population. Moreover,
Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides
are shown to directly interact in the yeast two-hybrid system. A
site-directed mutagenesis was performed on the SLS1 coding
sequence that allowed us to define a functional domain in Sls1p.
Indeed, co-translational translocation of a reporter protein is
affected when one of these mutant proteins is expressed. Moreover, this
protein has lost its capacity to interact with Kar2p, and the two
lumenal polypeptides might thus cooperate to promote secretory protein
co-translational translocation.
To initiate their pathway, secretory proteins are first targeted
to the endoplasmic reticulum
(ER)1 membrane in eukaryotic
organisms. In mammalian cells, a cytoplasmic particle (the
Signal Recognition Particle or SRP)
first recognizes these proteins when the signal peptide emerges from
the ribosome and then ensures their delivery to the ER membrane through
its interaction with the SRP receptor (1). SRP binding to the nascent chain-ribosome complex causes a translational pause that is released upon docking of SRP (2). As translation resumes, the complex is
transferred to the translocation site where crossing through the ER
membrane takes place. Sec61 In the model yeast, Saccharomyces cerevisiae, components
involved in SRP-dependent targeting are not essential, and
several secretory proteins were shown to cross the ER membrane
post-translationally (5, 6). This translocation mode relies both on
cytosolic chaperones whose binding delays preproteins folding (7) and on membrane proteins that ensure specific insertion of secretory proteins at the translocation site. A heptameric complex containing the
trimeric Sec61 complex and four other polypeptides, Sec62p, Sec63p,
Sec71p, and Sec72p, allows in vitro post-translational translocation of several preproteins in the presence of the lumenal chaperone Kar2p and ATP (8). All these polypeptides were previously identified in genetic screens and through biochemical approaches (9,
10). A contribution of proteins of the Hsp70 family has been described
for two preprotein transport machineries. The Kar2p and mitochondrial
matrix mHsp70 proteins, respectively, promote translocation across the
endoplasmic reticulum membrane and across the mitochondrial inner
membrane in yeast. While the transmembrane protein Sec63p was
identified as the Kar2p-binding partner in the ER membrane (11), two
components of the Tim complex, Tim44 and Tim17, were found to function
as membrane anchor for mHsp70 (12, 13).
In the yeast Yarrowia lipolytica, inactivation of the two
genes encoding the 7 S RNA component of the SRP is lethal (14), whereas
deletion of the SRP54 and SEC65 genes result in
very low growth (15). SRP is also essential in the yeast
Schizosaccharomyces pombe (16). Study of Y. lipolytica conditional lethal 7 S RNA mutants provided the first
in vivo evidence of SRP involvement in the targeting step of
the co-translational translocation process (17). Pursuing this genetic
approach, secondary mutations that led to synthetic lethality in
combination with the 7 S RNA mutation were selected. This screen
allowed cloning of the SLS1 gene whose product, located in
the lumen of the endoplasmic reticulum, was shown to participate in
preprotein translocation (18). To gain insights in Sls1p function,
putative associations with known ER resident proteins were tested. In
the present paper, we show that Sls1p directly interacts with the
chaperone protein Kar2p. We also show by a fractionation procedure that
the majority of translocation pores in Y. lipolytica are
linked to ribosomes and that Sls1p and Kar2p partially
co-fractionate with ribosome-bound Sec61p. To investigate the
structure/function relationship, we constructed different Sls1p mutant
proteins and compared their ability to interact with Kar2p with their
capacity to promote secretory protein translocation. One of these
mutant proteins, Sls1 Strains and Plasmids--
Plasmid pINA1077 contains a 2.2-kb
ClaI-SalI fragment comprising the entire
SLS1 open reading frame inserted into the multicloning site
of the phagemid Bluescript SK
Plasmid pAS2 Antibodies--
The 17 C-terminal amino acid residues from
YlSec62p were replaced by a peptide corresponding to the 13 C-terminal
amino acid residues of ScSec62p, allowing detection of the Y. lipolytica Sec62p using anti-S. cerevisiae Sec62p
antibodies. ScSec62p and YlKar2p antibodies were kind gifts of R. Schekman and of D. Ogrydziak, respectively. Anti-YlSec61p were raised
against the 14 N-terminal amino acid residues and were purified on
protein A-Sepharose as described in Görlich and Rapoport (4).
Preparation and Differential Extraction of Microsomes from
Yarrowia lipolytica--
Yeast cells grown in rich medium and
harvested during exponential phase were lysed by a 10-min vortexing
with glass beads in homogenizing buffer (250 mM Hepes-KOH,
pH 7.5, 25 mM potassium acetate, 5 mM magnesium
acetate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 50% glycerol). Supernatant from a low speed
centrifugation was submitted to a 30-min centrifugation at 15,000 rpm.
The resulting microsomal pellet was resuspended in homogenizing buffer
at a final concentration of 1-2 equivalents per microliter (1 equivalent is defined as 1 microliter of a membrane suspension with an
absorbance at 280 nanometers of 50). 100 equivalents were then diluted
in 500 microliters of 2× solubilizing buffer (25 mM
Hepes-KOH, pH 7.5, 400 mM potassium acetate, 8 mM magnesium acetate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol),
detergents were added, and samples were incubated for 30 min on ice. A
40-min centrifugation at 70,000 rpm in a 100.4 rotor in a Beckman
tabletop ultracentrifuge then yielded a supernatant, containing
solubilized material, and a ribosomal pellet. After resuspension in a
high salt buffer containing 2 mM puromycin and 0.2 mM GTP, the pellet was incubated for 30 min on ice and then
for 30 min at 28 °C. Ribosome-associated membrane proteins (RAMPs)
were detected in the supernatant of a 1-h centrifugation at 100,000 rpm
in a 100.4 rotor in a Beckman tabletop ultracentrifuge.
Binding to Concanavalin A-Sepharose, Immunoprecipitation, and
Western Blotting--
Supernatant fractions and pellet samples were
diluted in 500 microliters of 20 mM Hepes-KOH, pH 7.5, 150 mM potassium acetate, 5 mM magnesium acetate, 1 mM phenylmethylsulfonyl fluoride buffer before addition
either of 50 microliters of concanavalin A-Sepharose or 100 microliters
of anti-YlSec61p antibodies and overnight incubation at 4 °C. For
Kar2p immunoprecipitation, microsomes in solubilizing buffer were
incubated for 30 min on ice in the presence of 2% Triton X-100.
Unsolubilized material was discarded by a 30-min centrifugation at
15,000 rpm in a 100.4 rotor in a Beckman tabletop ultracentrifuge, and
anti-Kar2p antibodies plus protein A-Sepharose were added to the clear
supernatant after a 4-fold dilution in cold phosphate-buffered saline.
Immunoprecipitation was performed overnight at 4 °C. Sepharose beads
were washed three times with 500 microliters of phosphate-buffered
saline, and precipitates were eluted in sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 3% SDS, Bromphenol
blue 0.02%) for 20 min at 55 °C. Samples were then applied on a
10% polyacrylamide denaturant gel, and proteins were transferred onto
nitrocellulose membrane after the migration. Sec61p, Sec62p, Kar2p, and
Sls1p were revealed using specific polyclonal antibodies as primary
antibodies and peroxidase-conjugated anti-IgG antibodies as secondary
ones. Detection was realized using the ECL method (Amersham Pharmacia Biotech).
Microsomes Solubilization and Translocation Pores
Fractionation--
To gain insights about the nature of the
translocation sites in Yarrowia lipolytica, we checked for
the distribution of the major component of the translocation pore,
Sec61p, between ribosome-bound and free subcomplexes in comparison with
S. cerevisiae (8). The first step of this fractionation
experiment consists in solubilization of Y. lipolytica rough
microsomes by detergent. For this purpose, three different detergents
were used, and the behavior of YlSec61p as a marker of all
translocation pores and of YlSec62p as a marker of the
post-translational translocation complex was followed using specific
antibodies. A membrane-enriched pellet resuspended in 800 mM KAc was treated either with 2% digitonin, or with 2 and 4% Triton X-100, or with 1 and 2% Sls1p and Kar2p Associate with Sec61p in the Ribosomal
Pellet--
Since Sls1p and Sec61p were previously shown to
co-precipitate (18), the distribution of Sls1p in the same
fractionation procedure was tested using specific antibodies. We also
checked for the presence in the different fractions of the major
lumenal protein Kar2p that was shown to participate in the
translocation processes in S. cerevisiae (8, 21) and to seal
the translocation pore at an early step of co-translational
translocation (22). Microsomes solubilization was performed in the
presence of 2% Triton X-100 in 400 mM KAc, unsolubilized
material was discarded after a low speed centrifugation, and
supernatant was then submitted to a 70,000 rpm centrifugation, yielding
a high-speed supernatant and a ribosomal pellet. Each fraction was then
incubated in the presence of either concanavalin A-Sepharose or
anti-YlSec61p antibodies coupled to protein A-Sepharose. While the
majority of these two ER lumenal proteins was recovered in the
supernatant fraction as expected for soluble proteins, significant
amounts of the two proteins were also detected in the ribosomal pellet
(Fig. 2A, lane 1).
The following data strongly suggest that these two polypeptides are
specifically associated to the ribosome-linked YlSec61p population and
do not represent unspecific contamination. First, 40 to 50% of Sls1p
and Kar2p present in the pellet fraction were bound by concanavalin
A-Sepharose (lane 2). Because neither Sls1p nor Kar2p are
glycosylated (15), this suggests that they belong to a complex containing at least one glycosylated partner. Second, both were co-immunoprecipitated with YlSec61p (lane 3). Third, the
ratio of the two proteins in these two samples and in the ribosomal fraction was conserved (lanes 2 and 3 compared
with lane 1). Sls1p and Kar2p association with the
ribosome-linked Sec61p subcomplex in Y. lipolytica argues
for a participation in the co-translational translocation process. A
co-immunoprecipitation experiment was then performed to confirm that
Sls1p and the chaperone protein Kar2p belong to the same sub-complex.
Anti-Kar2p antibodies were added to a microsomal extract, and
immunoprecipitates were resolved on SDS-PAGE and blotted with
polyclonal anti-Sls1p. As shown in Fig. 2B, a Sls1p signal
was detected in the Kar2p-immunoprecipitate (lane 2). This
signal was absent when precipitation was performed with the pre-immune
serum (data not shown). To address the question of whether the two
proteins directly interacted in vivo, we used the S. cerevisiae two-hybrid system (23). Kar2p and Sls1p coding sequences were cloned in-frame in each of the two vectors. Results are
presented in Fig. 3. While all controls
were negative (sectors 1, 2, and 3), co-expression of the GAL4-BD-Sls1p
and GAL4-AD-Kar2p (sector 4) gave a positive result, allowing
transformants to grow on medium devoid of adenine (Fig. 3B)
or on medium devoid of histidine in the presence of 3-aminotriazole
(Fig. 3C) and to increase Temperature-sensitive Growth Phenotype Complementing Activity of
Sls1p Mutant Proteins--
To define Sls1p domains involved in partner
binding and to gain insights into Sls1p function, we constructed mutant
proteins by site-directed mutagenesis of the SLS1 gene.
Research of an open reading frame sharing some homology with Y. lipolytica Sls1p in the S. cerevisiae genome data base
revealed one putative homolog that displays 52% similarity and 27.5%
identity and contains the two N- and C-terminal ER localization signals
(Fig. 4). Four mutations (1, 2, 4, and 5)
were designed in which the most conserved regions between the
Y.l product and the S. cerevisiae protein were
deleted (see Table I and Fig. 4).
Mutation 6 consists of substitution of the arginine at position 222. To
test the effects of these mutations, the five mutated sequences were
integrated at the LEU2 locus of the Secretion Phenotype Displayed by sls1 Mutants and Kar2p-binding
Property of Sls1p Mutant Proteins--
To further characterize
sls1 mutants, their secretion phenotype was studied. In a
first approach, synthesis and/or secretion of an active alkaline
extracellular protease (AEP) was estimated on skim milk plates. After a
48-h incubation at 30 °C, halos were formed around the growing
colonies when all mutated alleles, except sls1 We defined solubilization conditions for Y. lipolytica
microsomes that allowed us in a subsequent fractionation experiment to
show that the major translocation pore component, Sec61p, distributes between two sub-complexes: one not linked to ribosomes and the other
associated to membrane-bound ribosomes as in S. cerevisiae (8). The two yeasts differ in the respective ratio of these two
populations. Indeed, from 70 to 80% of Sec61p is present in the RAMP
fraction in Y. lipolytica, whereas this ratio drops to 30%
in S. cerevisiae. Sec62p, which belongs to the membrane
protein complex required for post-translational translocation (8), is
preferentially recovered in the first subcomplex and is absent from the
RAMP fraction, indicating that this fraction contains translocation
complex involved in co-translational translocation events. In these
conditions, small amounts of the chaperone protein Kar2p co-fractionate
with Sec61p in the ribosomal pellet, suggesting that the two proteins
are associated in the same sub-complex. Accordingly, Kar2p present in
this fraction is co-immunoprecipitated by Sec61p antibodies.
Involvement of Kar2p in co-translational translocation was already
described in the yeast S. cerevisiae (21), and the chaperone
protein functions as a mammalian translocation pore gate (22). We also
checked the behavior of a second lumenal protein identified in Y. lipolytica, Sls1p, and showed that minor quantities of this
protein are also associated to the ribosome-linked Sec61p population.
Using the two-hybrid system, we then showed that Sls1p directly
interacts with Kar2p and thus constitutes a new partner of this
chaperone protein.
A mutational analysis then allowed us to conclude that the peptide
sequence from amino acid residues 382 to 387 is required for Sls1p
function in co-translational translocation and for Sls1p interaction
with the chaperone protein Kar2p. The decrease of the co-translational
translocation rate in a sls1 Sls1p does not contain the 70-amino acid residues DnaJ-like domain
present in Sec63p, which represents the main partner of Kar2p in the
yeast ER membrane (11). However, the fact that Tim44 displays a very
short domain of homology with the Sec63 DnaJ-like domain, and that
Tim17 shares no homology at all with it (13), indicate that co-factors
of the Hsp70 protein family in translocation are not restricted to the
DnaJ protein family. Moreover, because the Sls1p-Kar2p complex does not
exist at a 1:1 ratio, we can speculate that Kar2p binds to at least a
second partner in the translocation complex. This partner could be a homolog of the S. cerevisiae Sec63p as the two proteins were
shown to be involved in co-translational translocation (21) or another membrane protein with regard to Kar2p function in sealing the translocation pore at an early stage of the co-translational
translocation process (22). Interaction of Sls1p with secretory protein
and dependence on ATP of the Sls1p-Kar2p complex function in
translocation will be tested in an in vitro translocation system.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, one of the three polypeptides of the
translocation pore, is a polytopic membrane protein; the two others,
Sec61
and 
, span the ER membrane once. Three to four units of
this hetero-oligomer are needed to form the aqueous pore (3). In
vitro reconstitution experiments, using purified mammalian
components, show that the SRP receptor and the Sec61 complex are
sufficient to achieve translocation of some preproteins, while the TRAM
protein is required for translocation of other preproteins (4).
In vivo, many other soluble or membrane proteins could be
involved in this process to adjust the translocation rate to cell growth.
5p, that does not further associate with Kar2p
confers a decrease in the synthesis of a secreted protein, suggesting
that such an interaction is required for efficient co-translational translocation.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(Stratagene) (18). Mutant
forms of this plasmid were obtained by in vitro
site-directed mutagenesis using the Kunkel method (19). Five
oligonucleotides were used as primers to introduce mutations or
deletions (see Table I), and the presence of the mutations was
confirmed by sequencing. The ClaI-SalI fragments, containing the deletions, were subcloned in a Y. lipolytica
integrative plasmid and integrated in the genome of the
sls1 strain previously constructed: MatB,
scr1::ADE1, SCR2, his-1,
leu2, ura3, sls1::URA3 (18).
(TRP1, Ampr) was used
for expression of GAL4-DNA binding domain fusion proteins and plasmid
pACT2 (LEU2, Ampr) for expression of
GAL4-activating domain fusion proteins. The S. cerevisiae
strain PJ694A (MATa, trp1901,
leu23, 112, ura352, his3200,
gal4, gal80,
LYS2::GAL1-HIS3, GAL2-ADE2,
met2::GAL7-lacZ) was used for the two-hybrid
analysis (20). Two couples of primers were designed to amplify the
KAR2 nucleotide sequence between codon 27 and codon 656 (Kar2p-1: 5'-CCG GCC ATG GGC GTT CAG GCT GAT GAC GTG, and
Kar2p4: 5'-CGG GAT CCC ACC GTC GTT GGA CTC GTC TC) and the
SLS1 coding sequence from codon 19 to codon 411 (Sls1-:
5'-CCG GCC ATG GGC GAG GAT GAA ATC TGC AGA and Sls1-:
5'-CGG GAT CCA TAA CTC GCC TCG GTC CTG). After
amplification, polymerase chain reaction products were digested by
NcoI and BamHI to allow in-frame cloning in the two-hybrid vectors multicloning site. Empty and recombinant two-hybrid vectors were co-transformed into yeast cells by the lithium acetate method, and transformants were selected on minimal medium supplemented with histidine, methionine, uracil, and adenine for selection of the
plasmids and on minimal medium supplemented with histidine, methionine,
and uracil, or on minimal medium supplemented with adenine, methionine,
and uracil plus 5 mM 3-aminotriazole for direct selection
of the interaction (20).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-octoglucoside. After
sedimentation of ribosomes (P fraction), supernatants corresponding to
solubilized extracts were incubated in the presence of concanavalin
A-Sepharose, which retains glycosylated proteins (CA fraction), and
unbound material was then trichloroacetic acid-precipitated (U
fraction). Solubilization of S. cerevisiae microsomes was
performed as described in Görlich and Rapoport (4). No YlSec61p
and only small amounts of YlSec62p were released from the ribosomal
pellet using 2% digitonin, whereas about 70% of ScSec61p was
solubilized under these conditions (data not shown). Microsomes were
also not solubilized using -octoglucoside. However, as shown in Fig.
1A, 4% Triton X-100 led to
efficient solubilization of membrane proteins because almost all
YlSec62p was present in the supernatant fraction (lanes 1 and 2). About 80% of solubilized YlSec62p sedimented with
concanavalin A beads (lane 2 compared with lane
1), suggesting that this YlSec62p population belongs to a membrane
subcomplex containing at least one glycosylated protein. Under these
conditions, only 25% of YlSec61p was released from the ribosomal
pellet (lane 2 compared with lane 3), and all of
the solubilized YlSec61p was bound by concanavalin A beads (lane
2 compared with lane 1). In contrast, only 50% of
ribosome-free ScSec61p, which represents two-thirds of the total
ScSec61p population, binds to the lectin (data not shown). The
solubilization efficiency and YlSec61p distribution were almost the
same using a Triton X-100 concentration of 2% and a KAc concentration
of 400 mM (data not shown). To confirm that YlSec61p
remaining in the pellet fraction corresponded to ribosome-bound
material, puromycin was added to this fraction and salt concentration
was increased to release the RAMPs (4, 8). In this experiment,
microsomes were first solubilized using 4% Triton X-100 in 400 mM KAc, and salt concentration was then shifted to 800 mM. Concanavalin A-Sepharose was added to the supernatant
fraction of a 100,000 rpm centrifugation. As shown in Fig.
1B, YlSec61p was absent from the pellet fraction after
separation of the ribosomal subunits (lane 3 compared with 2), and all released YlSec61p sedimented with the lectin
(lane 5 compared with 4). About 75% of Sec61p
thus co-fractionates with ribosomes in Y. lipolytica in
contrast with S. cerevisiae, where only 30% of Sec61p was
present in the RAMP fraction (data not shown).
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Fig. 1.
Solubilization and fractionation of Y. lipolytica endoplasmic reticulum membrane protein.
A, solubilization of Y. lipolytica microsomes. 20 equivalents were resuspended in a solubilizing buffer containing 50 mM Hepes-KOH, pH 7.5, 800 mM potassium acetate,
16 mM magnesium acetate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 4%
Triton X-100. After a 30-min incubation on ice, the sample was
centrifuged for 40 min at 70,000 rpm. Supernatant fractions were
incubated in the presence of 50 microliters of concanavalin
A-Sepharose. Trichloroacetic acid-precipitated unbound material
(lane 1), lectin-bound material (lane 2), and
pellet fractions (lane 3) were incubated in sample buffer
(100 mM Tris-HCl, pH 6.8, 20% glycerol, 3% SDS, 0.02%
Bromphenol blue) for 20 min at 55 °C before SDS-PAGE analysis and
blotting using specific polyclonal anti-Sec61p and Sec62p antibodies.
B, fractionation of the ribosome-associated membrane
proteins. A ribosomal pellet was resuspended in a high salt buffer
containing 2 mM puromycin and 0.2 mM GTP and
incubated for 30 min on ice and then for 30 min at 28 °C before a
1-h centrifugation at 100,000 rpm yielding a pellet fraction
(lane 2) and the RAMP fraction (lane 3). The
latter fraction was incubated with concanavalin A-Sepharose, and bound
(lane 4) and unbound material (lane 5) were
analyzed by SDS-PAGE. Proteins were immunoblotted with anti-Sec61p
antibodies. Lane 1, crude microsomes.
-galactosidase expression
15-fold (data not shown).
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Fig. 2.
Kar2p and Sls1p association.
A, 20 equivalents of Y. lipolytica microsomes
were solubilized using 2% Triton X-100 in a potassium acetate
concentration of 400 mM. Supernatant from a low speed
centrifugation was then submitted to a 70,000 rpm centrifugation
yielding a ribosomal pellet (lane 1). Pellet samples were
incubated either in the presence of 50 microliters of concanavalin
A-Sepharose (lane 2) or 100 microliters of anti-YlSec61p
antibodies (lane 3). Precipitates were eluted in sample
buffer for SDS-PAGE analysis. Proteins were immunoblotted with
anti-Sls1p and Kar2p antibodies. B, anti-Kar2p antibodies
were added to solubilized material from a microsomal extract. Kar2p
immunoprecipitates were then resolved by SDS-PAGE, transferred to
nitrocellulose, and blotted with anti-Sls1p antibodies (lane
2) and compared with Sls1p detected in crude extract (lane
1).
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Fig. 3.
Kar2p and Sls1p interaction in the yeast
two-hybrid system. PJ69 4A strain was co-transformed with the
following plasmid combinations: 1, pAS2 and pACT2;
2, pAS2 and pACT2-Kar2p; 3,
pAS2-Sls1p and pACT2; 4, pAS2-Sls1p and
pACT2-Kar2p; 5, pAS2-Sls15p and pACT2-Kar2p, and
then plated on minimal medium lacking leucine and tryptophan
(A); minimal medium lacking leucine, tryptophan, and adenine
(B) and minimal medium lacking leucine, tryptophan, and
histidine with 5 mM 3-aminotriazole (C).
sls1
strain. Because the null strain was previously shown to display a
strong temperature-sensitive growth phenotype (18), Leu+
transformants were grown on YPD plates at 32 °C and compared with a
wild-type transformant. Only sls14 clones were unable to
grow at this temperature. All other mutated alleles restored a wild
type growth (data not shown). To address the question of whether equal
amounts of the mutant proteins were made in each strain, an
Sls1p-immunoblot analysis was performed. Although the same amount of
total intracellular protein was applied on the Western blot, no signal
corresponding to Sls14p was detected (data not shown). Deletion of
the LRNNP sequence thus appears to confer unstability to the mutant
protein. Substitution of the conserved basic residue of this peptide
for a neutral one had no effect on the protein stability since Sls16p
was as abundant as the wild type protein and conferred no visible
phenotype (data not shown).
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Fig. 4.
Alignment of Y. lipolytica Sls1p
with its S. cerevisiae homolog (YOLO31C). Substituted
and deleted amino acid residues are overlined.
Construction of five mutated versions of Sls 1p
4 and
sls15, were expressed. A small area of hydrolysis was
detected around the colonies of these two mutants after an additional
24-h incubation at the semi-permissive temperature of 28 °C (data
not shown). To know if the deficit of protease activity in the
sls15 mutant strain was because of a defect in AEP
translocation, a pulse-chase labeling experiment was performed after a
10-min shift at 30 °C (18). The kinetic of appearance of the AEP
intracellular mature form in this strain was similar to that of the
wild type strain as shown in Fig. 5,
indicating that translocated polypeptides are efficiently processed in
the sls15 mutant. AEP intracellular precursors were
detected immediately for the two strains. The only difference resides
in the amount of newly synthesized AEP precursors: about two-fold less
counts were present in the precursor form in an sls15
context compared with the value obtained for the SLS1 strain
(Fig. 5) while incorporation in total protein was similar. This result
indicates that the translocation rate of this co-translationally
translocated reporter protein is decreased in the mutant strain. To
correlate the secretion phenotype conferred by the Sls15p mutant
protein to a potential modification of its capacity to interact with
the chaperone protein, a co-immunoprecipitation experiment was
performed (Fig. 6). While Kar2p amounts
were similar in the two strains (lanes 1 and 2), no significant amount of Sls15p co-precipitating material was detected in the Kar2p immunoprecipitate compared with the wild type
protein (lane 6 compared with lane 4). To confirm
this first result, the mutated sequence was cloned in the two-hybrid
plasmid pAS2 and tested against the GAL4-AD-Kar2p fusion protein.
No association of the Sls15p mutant protein with Kar2p was detected in this test as shown in Fig. 3 (sector 5), although
expression levels of the fusion protein were similar to that of the
wild type Sls1p hybrid product (data not shown). Moreover, the
Kar2p-binding property of other mutant proteins, which fully
complemented growth and secretion defects of the null strain, was
detected using this in vivo method (data not shown).
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Fig. 5.
Secretion phenotype displayed by the
sls1 5 mutant. Radioactive counts measured for the
wild type and sls15 strains in the two AEP intracellular
forms at the different time points of the pulse-chase labeling
experiment. SLS1 and sls15 strains were grown
overnight in inducible medium, concentrated, and transferred at
30 °C for 10 min before the 45 s labeling in the presence of a
[35S]methionine and cysteine mix. Chase was performed by
addition of an excess of cold methionine and cysteine, and samples were
taken immediately and 1.5, 3, 5, and 10 min after. Cells were
sedimented, and intracellular proteins were immunoprecipitated by
anti-AEP antibodies. Immunoprecipitates were resolved on a denaturant
12% polyacrylamide gel and then dried and fluorographed. Solid
diamond, SLS1-pAEP; gray box,
sls1-5-pAEP; gray diamond, SLS1-mAEP;
×, sls1-5-mAEP.
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Fig. 6.
Kar2p-binding property of the Sls1 5p mutant
protein. 200 microliters of solubilized extracts from
SLS1 and sls15 strains were diluted in
phosphate buffer saline plus 1 mM phenylmethylsulfonyl
fluoride for immunoprecipitation in the presence of anti-Kar2p
antibodies and protein A-Sepharose for 4 h at 4 °C. After
washes, immunoprecipitates (IP) were eluted at 65 °C in
sample buffer and resolved on SDS-PAGE with crude extracts
(CE). Proteins were then transferred to nitrocellulose
membranes and blotted with anti-Kar2p (lanes 1 and
2) and anti-Sls1p antibodies (lanes 3 to
6). Lanes 1, 3, and 4, SLS1 strain; lanes 2, 5, and
6, sls15 strain; Ab, antibodies chains.
DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
5 context is not linked to a
temperature-sensitive growth phenotype as in the sls1
strain, suggesting that the Sls15 protein still retains a partial
function, which is independent of Kar2p-binding. Accordingly, the delay
in the secretion of the alkaline extracellular protease and the
induction of Kar2p expression observed in the absence of Sls1p (18)
were not further detected in a sls15 context. Studying the
mutant and the protein characteristics allows us to propose a model for
Sls1p function in the translocation process: Sls1p could first bind to
the incoming polypeptide at the lumenal side of the translocation pore.
The preprotein could then be directly transferred to the chaperone
protein, Kar2p that is recruited to the translocation site through its
association with Sls1p, facilitating in that way the crossing of the
preprotein. Alternatively, Sls1p could be anchored to the translocation
complex through its interaction with Kar2p that thus first binds to the
preprotein and then transfers it to Sls1p. Sls1p and Kar2p could act as
molecular ratchets as has been proposed for Tim44 and mHsp70 (13). Such an active mechanism could concern a subset of co-translationally translocated secretory proteins; for the majority of them, elongation on the ribosomes may be sufficient to ensure their transfer across the
ER membrane (24). In the presence of Sls15p, the transfer of the
incoming preprotein from one partner to the other would be delayed
because Sls1p-Kar2p interaction is inhibited. This study thus provides
evidence of Sls1p involvement in the co-translational translocation
process that relies on its association with Kar2p.
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ACKNOWLEDGEMENTS |
|---|
We thank M. Fromont for the PAS2
plasmid and P. James for the PJ69-4A strain and discussion.
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FOOTNOTES |
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* This work was supported by European Community Contract BIO2-CT96-003, by grants from INA P-G, INRA, and CNRS in France, and by grants from DFG in Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax:
33-01-30-81-54-57; E-mail: boisrame{at}cardere.grignon.inra.fr.
The abbreviations used are: ER, endoplasmic reticulum; SRP, signal recognition particle; RAMP, ribosome-associated membrane protein; AEP, alkaline extracellular protease; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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