Departments of Microbiology and Molecular
Genetics and Biological Chemistry, University of California,
Irvine, Irvine, California 92697-1700
Received 2 March 2000/Returned for modification 18 April
2000/Accepted 25 May 2000
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INTRODUCTION |
Nucleocytoplasmic transport is a
complex process mediated by interaction of transport cargoes with
components of the nuclear pore complex along with other soluble factors
(reviewed in references 9, 28, and
30). Import of most karyophilic proteins into the
nucleus is energy dependent and mediated by short stretches of amino
acids known as nuclear localization signals (NLSs). Proteins with
classical NLSs, as exemplified by the simian virus 40 (SV40) large T
antigen NLS and the nucleoplasmin NLS, are transported to the
nucleus by a heterodimer composed of Srp1p (the Saccharomyces cerevisiae homolog of importin 
[Kap60p]) and Kap95p (an
S. cerevisiae homolog of importin 
) (6, 10).
Srp1p is the NLS receptor component that binds to the NLS itself, while
Kap95p interacts with some components of the nuclear pore complex to
facilitate movement of the Srp1p-Kap95p complex carrying a cargo
protein from the cytoplasm to the nucleus (35, 36, 37).
There are several other receptor proteins, which have amino
acid sequence similarity to importin (Kap95p) and are grouped in
the importin family. These proteins directly bind cargo proteins such as ribosomal proteins and carry out a protein import function independently of Srp1p (reviewed in references 9,
28, and 30).
SRP1 was originally identified as a suppressor of certain
temperature-sensitive (ts) mutations in RNA polymerase I (Pol I) in
S. cerevisiae (54). Specific mutations in
SRP1 were able to suppress ts mutations in the zinc-binding
domains of RPA190 and RPA135, genes encoding the
largest and second largest subunits of Pol I, respectively, but not
other rpa190 and rpa135 ts mutations outside of
this domain. These SRP1 suppressor mutations displayed no
apparent phenotype on their own. Additional work has shown that
different ts mutations in SRP1 cause different defects,
which include protein import defects, nuclear segregation defects,
altered microtubule morphology, and altered nucleolar morphology
(24, 44, 55). Although the role of Srp1p in protein import
is firmly established, it is not clear whether all of the diverse and
allele-specific phenotypes displayed by these mutations are the
consequences of defects in protein import. For example, while defects
in NLS binding and NLS protein import were clearly demonstrated for the
srp1-31 mutation (24, 44), no or only slight
defects in NLS binding and protein import were observed for the
srp1-49 mutation, which showed various other phenotypes
(44, 55; unpublished data). Likewise, suppression of
Pol I ts mutations by SRP1 mutations is difficult to explain
on the basis of the Srp1p function as the NLS receptor (54,
55).
For these reasons, we considered and tested the possibility that Srp1p
might have an additional function(s) distinct from the NLS receptor
function in protein import. We now report our discovery that two
mutations, srp1-49 and srp1-31, show intragenic complementation, indicating that the function affected by
srp1-49 is clearly different from the function affected by
srp1-31, i.e., the NLS receptor function. To obtain clues to
the functions affected by srp1-49, high-dosage suppressors
of srp1-49 were isolated. Two suppressors identified,
STS1 and RPN11, suppressed only
srp1-49 and not srp1-31. STS1 was
originally identified (22) as a dosage-dependent suppressor
of a mutation in SEC23, a gene required for endoplasmic reticulum (ER)-Golgi protein transport (56). Independently, DBF8, which is identical to STS1, was also
identified as a gene essential for nuclear segregation and division
(12). RPN11 was recently identified as a
component of the regulatory particle of the 26S proteasome
(8), suggesting a link between SRP1 and the
ubiquitin-proteasome system. Further experiments designed to examine
such a link have led to the conclusion that Srp1p, together with Sts1p,
carries out a function in the regulation of protein degradation by the
ubiquitin-proteasome system and this function is separate from its
established function in NLS-dependent protein import into the nucleus.
We report these experiments and also discuss previous observations
which support this conclusion.
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MATERIALS AND METHODS |
Media, strains, and plasmids.
YEPD medium consists of 2%
yeast extract, 1% Bacto-peptone (Difco Laboratories, Detroit, Mich.),
and 2% glucose. Synthetic glucose (SGlu) medium (2% glucose, 0.67%
yeast nitrogen base [Difco], and 0.5% Casamino acids [Difco])
was supplemented with required bases or tryptophan as described by
Sherman et al. (42). Synthetic galactose (SGal) and
synthetic raffinose (SRaff) media are the same as SGlu but with 2%
galactose or 3% raffinose substituted for glucose, respectively. To
make solid medium, 2% agar was added.
The yeast strains and plasmids used in this study are described in
Table 1. Yeast plasmid
transformations were performed as described by Gietz et al.
(7). DNA sequencing was performed using an ABI373A DNA
sequencer (PE Applied Biosystems). The SRP1 chromosomal
locus was replaced with the srp1-31 or srp1-49
mutant allele (55) by plasmid integration and pop-out
(24, 51).
The STS1 gene was amplified by PCR using lambda genomic
clone 70732 (American Type Culture Collection) as the template with the
primers 5' GGT ACA TAC TAG CGG CAG 3' and 5' CCG ACG ATG ACC ACT CAC
3'. The PCR product was cloned into the SmaI site of
pUC19, yielding pNOY3234. A SalI digest was performed
to remove STS1 and move it into the SalI site of
pRS314 and pRS316 (45), creating pNOY470 and pNOY471,
respectively. For STS1 genomic disruption, pNOY3234 was
digested with PflMI and NsiI to remove most
of the STS1 coding region, and this deletion was replaced
with the HIS3 gene to make pNOY3235. pNOY3235 was digested
with SalI, and the 1,934-bp fragment was gel isolated and
digested with PvuII. The SalI-PvuII
sts1::HIS3 fragment was then
transformed into NOY397, and His+ transformants were
selected (NOY726). NOY726 was sporulated, and tetrads were dissected
and showed a 2:2 segregation of His
to His+,
confirming that STS1 is an essential gene (12).
NOY726 was transformed with pNOY470 or pNOY471, and Trp+ or
Ura+ transformants were selected, respectively. Then the
strains were sporulated and tetrads were dissected to create NOY943 and NOY944.
Two-hybrid system plasmids were prepared as derivatives of pAS1 and
pACT2 (4). SFY526 (Clontech) was used as the reporter strain
for all two-hybrid experiments. pNOY472 was constructed by inserting
the NcoI-SacI and SacI-SalI
SRP1 fragments from pNOY3198 into the NcoI and
SalI sites of pAS1. STS1 was amplified using PCR
with the template lambda clone 70732 and the primers 5' CAT GCC ATG GGA
TTT GAA TGG GGT TTT AAA CCC 3' and 5' CGG AAT TCT TAG TTA AAG GGC GAA
TCA GTA G 3'. The PCR fragment was digested with NcoI and
EcoRI and cloned into the corresponding sites of pACT2
(pNOY473). The NcoI-XhoI fragment from pNOY473
containing the STS1 gene was ligated into the
NcoI and SalI sites of pAS1, creating pNOY474. An
RPN11-containing fragment, obtained by digesting pNOY285
with SalI, was treated with T4 DNA polymerase (New England Biolabs) to fill in the overhang and then SacI digested. The
blunt SacI fragment was ligated into the SmaI and
SacI sites of pACT2, creating pNOY475.
pNOY476 was constructed by cloning a NotI fragment
containing the open reading frame for green fluorescent protein (GFP)
(a gift from Pamela Silver) into the NotI site of pNOY477.
For constructing pNOY478, the GFP-STS1 fusion gene was
derived from pNOY476 as a KpnI blunted EagI
fragment. This fragment was ligated into pNOY258 previously
digested with BamHI, blunt ended with T4 DNA polymerase, and
then digested with EagI. pNOY479 was constructed by cloning the PflMI-BamHI fragment from pNOY476 into
pNOY471. pNOY480 was constructed using site-directed mutagenesis and
the oligonucleotide 5' CTT GCT CCT CGT TGG CGT AGA CTC CAG CAG TTG GAA
TC 3' to remove NLS1 (21). pNOY481 was constructed by
digesting pNOY258 with PflMI and SacII and
replacing the STS1 fragment with the sts1
NLS1 PflMI-SacII fragment from pNOY480. pNOY482 is a
derivative of pNOY478 made by exchanging the STS1 SacII
fragment for the sts1NLS1 SacII fragment from pNOY480.
pNOY483 is a derivative of pNOY258 but with E43G and I162T. pNOY484 was
constructed by moving the PflMI-SacI fragment
from pNOY483 into pNOY478 digested with PflMI and
SacI to remove STS1.
pNOY291 (glutathione-S-transferase
[GST]-STS1) was constructed by PCR using 5' TTT
GGA TCC CAT ATG ATG GGC TTT GAA TGG GGT TTT AAA CCG AGC AGC AAA AT 3'
and 5' CGC AAT TCT TAG TTA AAG GGC GAA TC 3' and pNOY258 as the
template. The fragment was digested with BamHI and
EcoRI and cloned into the BamHI and
EcoRI sites of pGEX2T (Pharmacia). pNOY3280 was produced
similarly but with pNOY480 as the template. pNOY488 was constructed by
ligating the 0.1-kb triple hemagglutinin epitope tag (HA-tag)
NotI fragment derived from the GTEP plasmid (39)
into the NotI site of pNOY478.
To construct the sts1(E43G) srp1-49 strain,
NOY514 was mated with NOY945. The diploids were cured of the wild-type
STS1 URA3 plasmid by streaking onto SGlu with 5-fluoroorotic
acid and the resulting strain was transformed with pNOY343, which
contains the sts1(E43G) mutation. The resulting diploid was
sporulated, and tetrads were dissected. Spores that were
Trp+, Leu+, His+, and
Ura+ were selected (NOY946).
For the experiments to study the stability of the
ubiquitin-Pro--galactosidase (Ub-P-gal), strains were
constructed by introducing plasmid pUb-P-ek-gal
(3) (obtained from A. Goldberg), carrying the gene for Ub-P-gal fused to a GAL promoter, into the strains to be tested.
NLS peptide binding assay.
The NLS peptide binding assay was
performed as described by Shulga et al. (44).
Protein pull-downs and immunoprecipitations.
GST fusion
proteins were produced and purified as described previously
(2). Srp1p was prepared from GST-Srp1p by thrombin cleavage,
and free GST was removed using glutathione agarose. For GST pull-down
reactions, 1 µg of input proteins was mixed together in 100 µl of
buffer M (25 mM ammonium sulfate, 50 mM Tris-HCl [pH 8.0], 5%
glycerol, 1 mM dithiothreitol, and 1 mM EDTA) and allowed to incubate
on ice for 30 min. Then, 200 µl of buffer M plus 0.2% bovine serum
albumin and 0.5% Tween 20 was added along with a 15-µl bed volume of
glutathione-agarose beads washed in buffer M, and the reactions were
incubated further for 45 min at 4°C with rotation. Beads were washed
four times with 400 µl of buffer M. Sodium dodecyl sulfate (SDS)
sample buffer was added to the beads, which were then boiled and
subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Proteins
were blotted onto Immobilon (Millipore, Bedford, Mass.), and the blot
was probed with sheep anti-Srp1 antibodies (54). SV40 large
T antigen NLS peptide and reverse large T antigen peptide were added to
reactions before incubations on ice and were described previously
(2).
For coimmunoprecipitation of HA-Sts1p and Srp1p, NOY944 and NOY947
cells were grown to an A600 of ~0.9 in 50 ml
of synthetic glucose medium. Cells were harvested by centrifugation and
washed with water. Extracts were prepared in buffer D (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM MgCl2, 1 mM
phenylmethylsulfonyl fluoride [PMSF], and 10% glycerol) by glass
bead lysis. The protein concentrations were determined using Bradford
reagent (Bio-Rad) and adjusted to be equal. Protein A-Sepharose and
Srp1 antiserum were then added to extracts (containing approximately 1 mg of protein) and mixed for 4 h at 4°C. The beads were then
washed with buffer A (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM EDTA,
1% Triton X-100), and samples were resolved on an SDS-10%
polyacrylamide gel and analyzed by Western blotting using anti-HA
antibody. The use of equivalent amounts of extracts in these
experiments was always confirmed by Coomassie staining of gels after
electrophoresis of samples.
Microscopy.
A Zeiss Axioplan 2 was used to visualize direct
and indirect fluorescence. For direct fluorescence of strains
containing galactose-inducible GFP constructs, strains were grown
overnight in SRaff medium. Production of GFP fusion protein was induced
by the addition of 3% galactose directly to the culture, and the cells
were observed 3 to 4 h after induction.
Degradation of -galactosidase test substrates.
Actively
growing cells (A600, <1.0) were harvested by
centrifugation and labeled in 200 µl of labeling buffer (50 mM sodium phosphate [pH 7.0], 2% galactose) with 0.25 mCi of
EXPRES35S protein labeling mix (NEN) for 5 min at 30°C.
Cells were then pelleted and resuspended in 400 µl of chase mix (2%
yeast extract, 1% peptone, 2% glucose, 10 mM Met, 5 mM Cys, and 0.6 mg of cycloheximide per ml), and incubation was continued at 30°C.
Aliquots were removed at specific time points and added to tubes
containing 400 µl of buffer A with protease inhibitors (aprotinin,
bestatin, pepstatin, leupeptin, and PMSF) and glass beads, and the
tubes were rapidly frozen. The cells were lysed by vortexing, and
radioactivity in trichloroacetic acid-precipitable material of the
extracts was determined. Equal counts were used for immunoprecipitation
reactions using anti--galactosidase antibodies (Promega) and protein
A-Sepharose (Pharmacia). The protein A-Sepharose beads were washed with
buffer A plus 0.1% SDS. Samples were analyzed by SDS-PAGE followed by autoradiography.
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RESULTS |
Intragenic complementation of srp1-31 and
srp1-49.
In previous work, ts defects in both in vitro NLS
peptide binding and in vivo NLS protein import were observed for the
srp1-31 mutation, whereas no or only very weak defects were
observed for the srp1-49 mutation (24, 44). In
order to test the possibility that these two mutations affected
separate functions of Srp1p, we carried out a complementation test.
Strains containing each of these two mutations on the chromosome were
constructed and transformed with CEN plasmids containing
SRP1, srp1-31, or srp1-49. The strains
were then streaked on selective medium at 38°C to check for
complementation of the ts growth phenotype by a different allele of
srp1.
Ts growth of the srp1-31 strain could be complemented by a
plasmid containing srp1-49, and ts growth of the
srp1-49 strain could be complemented by a plasmid carrying
srp1-31 (Fig. 1, sectors 956 and 959). Both mutant strains were complemented by the wild-type SRP1, confirming the recessive nature of the mutations, but
not by plasmids containing the identical mutation or by the vector plasmid (Fig. 1). These results indicate that the functions affected by
srp1-31 and srp1-49 are clearly different.
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FIG. 1.
Complementation of srp1-31 and
srp1-49 mutations. Strains carrying SRP1,
srp1-31, or srp1-49 on the chromosome were
transformed with plasmids containing SRP1,
srp1-31, or srp1-49. The resulting strains, as
listed in the figure, were streaked on synthetic glucose minus
tryptophan plates and incubated at the indicated temperatures for 3 days. Vector represents pRS314.
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Isolation of high-dosage suppressors of srp1-49.
In
order to understand the nature of the defect in the srp1-49
mutant, high-dosage suppressors of srp1-49 were isolated.
The srp1-49 strains NOY514 and NOY613 were transformed with
a galactose-inducible yeast cDNA library (23) and plated on
glucose selective medium at 25°C for 2 days. Transformants were then
replica plated onto galactose selective medium to induce expression of
the cDNAs and allowed to grow at 38°C for an additional 3 to 5 days.
Plasmid DNA was isolated from colonies that grew on galactose but not on glucose at 38°C, and DNA sequencing was performed to identify the
dosage-dependent suppressors. Two suppressors of srp1-49
were identified in this way, STS1 and RPN11 (Fig.
2A). Plasmids carrying the
STS1 or RPN11 suppressor were then transformed
into strains carrying the srp1-31 mutation. No suppression
was observed (Fig. 2B). Therefore, STS1 and RPN11
are allele-specific high-dosage suppressors of srp1-49.
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FIG. 2.
Dosage-dependent suppression of srp1-49 but
not srp1-31 by STS1 and RPN11. (A) The
srp1-49 strains containing vector only (pRS316-GAL), a
GAL-driven STS1 plasmid (pNOY258), and a
GAL-driven RPN11 plasmid (pNOY285) are shown
after growth on synthetic complete galactose (Gal) or glucose (Glu)
plates lacking uracil as indicated for 3 days at 25 or 38°C. (B) The
srp1-31 strains containing vector only, a
GAL-driven STS1 plasmid, and a
GAL-driven RPN11 plasmid were examined in the
same way. WT, wild type.
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Interaction of SRP1 with STS1.
We first
examined the possibility of whether the dosage-dependent
suppression of srp1-49 by STS1 reflected a
physical interaction of the two proteins encoded by these genes. An
interaction of the two proteins was first indicated by the yeast
two-hybrid system (Table 2). Second, we
detected in crude cell extracts a complex containing Srp1p and Sts1p by
coimmunoprecipitation. Extracts were prepared from a strain containing
HA-tagged STS1 (NOY947) and a control strain without the
HA-tag (NOY944). Srp1p was precipitated with Srp1p antiserum, and the
precipitates were subjected to SDS-PAGE followed by Western blot using
an anti-HA monoclonal antibody. As shown in Fig.
3A, HA-Sts1p was coimmunoprecipitated
with Srp1p (lane 5; lane 3 is a negative control using extracts without
Srp1p antiserum, which gave a very weak background signal). These two experiments strongly indicate that Srp1p and Sts1p interact in vivo.
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FIG. 3.
Interaction of Srp1p and Sts1p as demonstrated by
coimmunoprecipitation from cell extracts (A) and by the use of
GST-Sts1p fusion protein (B). (A) Extracts were prepared from the
strain carrying the HA-tagged STS1 gene (lanes 3 and 5) or
the control strain without the HA-tag (lanes 2 and 4). The extracts
were treated with anti-Srp1p antibodies (lanes 4 and 5) and protein
A-Sepharose, and precipitated proteins were subjected to SDS-PAGE
followed by Western analysis using a monoclonal anti-HA antibody to
detect HA-Sts1p. Lane 1 is an antibody control without extracts. Lanes
2 and 3 are negative controls without antibody. (B) Srp1p was incubated
with GST (lane 3), GST-Sts1p (lane 4), GST-Sts1NLS1p (lane 5),
GST-Sts1p plus 200 µM SV40 NLS peptide (lane 6), or GST-Sts1p plus
200 µM reverse NLS peptide (lane 7). Lane 2 did not receive any GST
fusion protein. GST or GST fusion proteins were pulled down using
glutathione-agarose beads and then subjected to SDS-PAGE followed by
Western blot using anti-Srp1p antibodies to detect Srp1p. Lane 1 is
10% of the input Srp1p preparation.
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In order to examine whether these two proteins interact directly
in vitro, a GST-STS1 fusion gene was constructed. Purified Srp1p and GST-Sts1p, which were both produced in Escherichia
coli as recombinant proteins, were mixed, and GST-Sts1p was
pulled down using glutathione-agarose. Pulled-down proteins were
separated by SDS-PAGE followed by Western blot analysis of Srp1p using
anti-Srp1p antibodies. As shown in Fig. 3B (lane 4), a direct
interaction between Srp1p and GST-Sts1p was indeed observed.
As described below, Sts1p is mostly localized to the nucleus. To
determine if the interaction between Srp1p and Sts1p could be through
an NLS in Sts1p, SV40 NLS peptide was added to the reaction mixture as
a competitor in the GST pull-down experiments. NLS peptide was able to
decrease the binding of GST-Sts1p to Srp1p (Fig. 3B, compare lane 6 to
lane 4), while a peptide containing the same amino acids in reverse
sequence was not (lane 7). Thus, the direct interaction of Srp1p with
GST-Sts1p in vitro appears to take place through an interaction similar
to that of Srp1p with classical NLS peptides.
Examination of the Sts1p sequence revealed two basic
regions in the amino-terminal end which could
potentially serve an NLS function, NLS1
(35KQKRR39) and NLS2
(58KYGGVSKRR66) (Fig.
4A). Both regions were individually
deleted using oligonucleotide-directed mutagenesis, and each deletion
was tested for complementation of the sts1 genomic deletion.
While the mutant carrying the deletion of NLS2 could still complement
an sts1 deletion strain, one carrying the deletion of NLS1
did not (data not shown). The production of Sts1NLS1 protein in
yeast cells was confirmed by Western blot analysis using anti-HA
antibody directed against an N-terminal HA epitope engineered into the
deletion protein construct. Therefore, the failure of
complementation was not due to lack of protein production. To further
characterize NLS1, a fusion protein containing the NLS1 deletion,
GST-Sts1NLS1p, was isolated from E. coli as a recombinant
protein and tested for its interaction with Srp1p in vitro. The
mutant fusion protein was unable to interact with Srp1p (Fig. 3B, lane
5). These experimental results strongly suggest that Sts1p carries an
NLS that resembles SV40 NLS and that the in vitro interaction of
GST-Sts1p with Srp1p is through this NLS-like sequence of Sts1p. Thus,
Sts1p may be a nuclear protein and its nuclear import from the
cytoplasm may involve its binding to Srp1p. However, based on the
results obtained by fractionation of cell extracts and by indirect
immunofluorescence microscopy, it was previously reported that Sts1p
was localized to the cytoplasm (1, 22). Therefore, the
question of localization of Sts1p was reexamined.
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FIG. 4.
Localization of Sts1p assayed by the use of GFP-Sts1
fusion protein. (A) Schematic representation of Sts1 protein, showing
locations of NLS1 and NLS2 as well as two point mutations, E43G and
I162T. (B) Strains carrying GFP-STS1,
GFP-sts1NLS1, and GFP-sts1-11,12(E43G, I162)
were grown in SRaff at 30°C, and synthesis of the fusion proteins was
induced by galactose. Localization of the protein was analyzed by
direct fluorescence microscopy. Nuclei were stained with
4',6'-diamidino-2-phenylindole (DAPI).
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Analysis of nuclear localization of Sts1p in the wild-type and
srp1 mutant strains.
We constructed a strain
containing a gene encoding a tagged GFP-Sts1 fusion protein under
control of the galactose promoter (NOY948) and analyzed localization of
the fusion protein after induction by galactose. As shown in Fig. 4B,
GFP-Sts1 showed a clear nuclear localization in vivo by its
fluorescence. This nuclear localization was also confirmed by indirect
immunofluorescence using anti-GFP antibodies with a strain containing
GFP-STS1 in an sts1 deletion background (data not
shown). Effects of the NLS1 deletion on protein localization were then
tested in vivo using a galactose-inducible GFP-sts1NLS1
fusion gene. Compared to the nuclear localization of GFP-Sts1p,
GFP-Sts1NLS1p showed a more cytoplasmic distribution (Fig. 4B). The
fact that GFP-Sts1NLS1p was not excluded from the nucleus may
suggest that there is another region in Sts1p with some NLS function.
Taken together, these data strongly suggest that Sts1p is mainly
localized to the nucleus and interacts with Srp1p via an NLS sequence
resembling the SV40 NLS sequence to gain entry into the nucleus.
To prove that Sts1p indeed uses Srp1p for nuclear import, a plasmid
containing galactose-inducible GFP-STS1 was transformed into
an srp1-31 ts strain, which shows defects in NLS-dependent protein import at nonpermissive temperatures, as mentioned above. Localization of GFP-Sts1p was then followed after a temperature shift
by direct fluorescence signal. GFP-Sts1p was found in both the nucleus
and the cytoplasm after 4 h at 37°C (data not shown) (48a). Therefore, Sts1p uses Srp1p, at least in part, to
gain entry into the nucleus. In contrast to the srp1-31
mutation, no defect in the localization of GFP-Sts1p in an
srp1-49 strain was observed during up to 8 h of
incubation at 37°C (data not shown) (48a), when many other
drastic mutational defects such as an alteration in the nucleolar
morphology had already taken place. Thus, suppression of
srp1-49 by a high dosage of STS1 is not due to compensation of a (hypothetical) decrease in the nuclear transport of Sts1p in srp1-49 mutants.
Interaction between Srp1p and Sts1p required for suppression
of srp1-49.
As described above, there is an interaction
of Sts1p with Srp1p through the NLS1 sequence of Sts1p. We
have found that this interaction may be prerequisite to suppression of
srp1-49 by STS1 but is not sufficient for
suppression. An additional interaction not involved in the protein
import function must take place for suppression. The mutant gene
sts1NLS1 was put under control of the galactose promoter
and transformed into the srp1-49 strain. Transformants were
then tested for their ability to grow at the nonpermissive temperature
in the presence of galactose. A high dosage of sts1NLS1
was unable to suppress the growth defect of srp1-49 at
38°C on galactose (Fig. 5, strain 965 compared to 963). The results show that direct interaction with Srp1p
through the NLS1 sequence may be required for a high dosage of
STS1 to suppress srp1-49. Since Sts1NLS1
protein is clearly present in the nucleus in addition to the cytoplasm,
as mentioned above (see Fig. 4B), it appears that the direct
interaction required for the suppression is probably not simply to
achieve nuclear transport of Sts1p. This inference was further
supported by the following experiments.
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FIG. 5.
Suppression of srp1-49 by a high dosage of
STS1 and sts1 mutants. Plasmids containing
STS1, sts1-11,12(E43G, I162T), and
sts1NLS1 under control of the GAL promoter
were transformed into the srp1-49 strain. Resulting strains
and a control SRP1 strain were streaked onto synthetic
complete galactose (Gal) or glucose (Glu) plates lacking uracil and
incubated for 3 days at 38°C. Vector pRS314 was used as a plasmid
control.
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We isolated sts1 mutants that did not show an interaction
with SRP1 in the two-hybrid assay. These mutants were then
screened for loss of the ability to suppress srp1-49 under
high-dosage expression. In this way, we found that an sts1
mutation containing a two-amino-acid substitution,
sts1-11,12(E43G I162T), abolished both the ability to
interact with SRP1 in the two-hybrid system (Table 2) and
high-dosage suppression of srp1-49 (Fig. 5, strain 961; for
the locations of the mutations, see Fig. 4A). We then examined whether
this double mutation inhibited the nuclear localization of Sts1p. Like
the wild-type GFP-Sts1p, the mutant GFP-Sts1(E43G, I162T)p was found to
be localized to the nucleus (Fig. 4B). Thus, Sts1p localization to the
nucleus is not sufficient for suppression of srp1-49. An
additional interaction between Sts1p and Srp1p, which is detected by
the two-hybrid system and is abolished by the E43G I162T double
mutation, appears to be required for suppression of srp1-49.
This additional interaction may or may not be a direct interaction and
most likely takes place within the nucleus.
Combination of the sts1-11 and srp1-49
mutations.
The above sts1 double mutation,
sts1-11,12, showed good complementation of growth
defects caused by an sts1 deletion; no difference was
observed in growth rate from the wild-type control (data not shown). Thus, the interaction disrupted by the double mutation and
required for suppression of srp1-49 does not appear to be essential for normal growth in the context of the wild-type
SRP1.
We also separated the two mutations, E43G and I162T, contained in the
sts1 double mutant sts1-11,12 and examined them
individually for the ability to interact with SRP1 in the
two-hybrid system and for the ability to suppress srp1-49 by
overexpression. Neither one of the mutations showed an interaction with
SRP1 in the two-hybrid system, and each partially abolished
the suppression of srp1-49 (data not shown). However,
in the course of these experiments, we found that although the
sts1-11(E43G) mutation itself did not show any growth defect
phenotype, a strain which contained both srp1-49 and
sts1-11 showed more severe temperature sensitivity than with
srp1-49 only (Fig. 6). This
result supports the above-described conclusion that the interaction of
Sts1p and Srp1p is functionally important; a point mutation that
abolishes this interaction in the two-hybrid system affects cell growth
at high temperatures in the context of the srp1-49 mutation.
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FIG. 6.
Temperature sensitivity of the sts1-11(E43G)
srp1-49 double mutant. Strains containing combinations of
wild-type STS1 or sts1(E43G) and wild-type
SRP1 or srp1-49 were streaked onto synthetic
complete glucose plates lacking tryptophan and uracil and grown for 3 days at 25, 36, or 38°C.
|
|
Interaction of STS1 and RPN11.
RPN11,
the other high-dosage suppressor of srp1-49, did not show an
interaction with SRP1 when tested by the two-hybrid system. However, it did show a positive interaction with STS1 in the
two-hybrid system (Table 2). This result suggests that STS1
and RPN11 interact either directly or indirectly
through other components and that this interaction might be related to
RPN11 suppression of srp1-49.
Degradation of Ub-P-gal in srp1-49 and
srp1-31.
The discovery that one of the high-dosage
suppressors of srp1-49 was RPN11, a subunit of
the 19S proteasome regulatory particle, led us to examine
ubiquitin-dependent protein degradation in srp1-49 cells.
For this purpose, we used Ub-P-gal, a model substrate of the
ubiquitin-proteasome pathway studied by previous investigators (3,
13). A plasmid carrying the gene for Ub-P-gal fused to a
GAL promoter was introduced into an srp1-49
strain, an srp1-31 strain, and a control wild-type
SRP1 strain, as well as srp1-49 strains carrying
an RPN11 or STS1 dosage suppressor plasmid.
Degradation of Ub-P-gal was then determined by pulse-chase and
immunoprecipitation analysis (3, 13).
As shown in Fig. 7A and B, Ub-P-gal
was substantially more stable in srp1-49 than in the control
strain, but no such stabilization was observed for srp1-31
(Fig. 7A). Significantly, both srp1-49 strains carrying the
suppressor RPN11 and the suppressor STS1 showed a
faster degradation rate approaching that shown by the control
strains (Fig. 7B), although the amounts of radioactive Ub-P-gal
synthesized during the pulse-labeling period were reduced in
these suppressed strains. We conclude that the srp1-49
mutation and its suppression by a high dosage of RPN11 or
STS1 are correlated with a decrease in the rate of
Ub-P-gal degradation and its restoration, respectively. The results
also demonstrate that the srp1-49 and srp1-31
mutations are clearly different in their effects on Ub-P-gal degradation, confirming that the two mutations affect different functions of Srp1p. We note that the experiments shown in Fig. 7 were
done at 30°C, which is a permissive temperature for these mutants. Similar results were also obtained when Ub-P-gal
degradation was assayed after a temperature shift from 30 to 38°C
(data not shown). Thus, defects in the ability to degrade Ub-P-gal
observed in srp1-49 do not affect growth at 30°C but
appear to cause growth defects at higher temperatures.
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FIG. 7.
Degradation of Ub-P-gal. Cells were grown overnight
in SGal minimal medium at 30°C and labeled with 35S
labeling mix for 5 min. The cells were resuspended in
cycloheximide-containing chase mix, and aliquots were removed at the
indicated times. The amount of labeled Ub-P-gal in the extracts was
analyzed by immunoprecipitation with anti--galactosidase antibodies
followed by SDS-PAGE and quantification by phosphorimager analysis. (A)
Degradation of Ub-P-gal in wild-type (WT), srp1-49, and
srp1-31 mutants (strains NOY934, NOY937, and NOY940,
respectively). (B) Degradation of Ub-P-gal in srp1-49
mutant in the presence of the suppressors RPN11 (NOY938) and
STS1 (NOY939). Degradation of Ub-P-gal was also assayed
simultaneously in srp1-49 in the presence of vector (NOY937)
and in wild-type cells (NOY934) as a control. The quantification of
Ub-P-gal is shown in the graphs below the autoradiograms.
|
|
Degradation of Ub-P-gal involves the initial ubiquitination
steps of the ubiquitin fusion degradation (UFD) pathway
which are different from other ubiquitination pathways, e.g., the
N-end rule pathway (13, 14, 16). Therefore, we tested
degradation of another model substrate, Leu-gal, which is degraded
through the N-end rule pathway (3). We observed that
Leu-gal degradation was also significantly slower in
srp1-49 than in the wild type (data not shown). Therefore,
defects caused by the srp1-49 mutation appear not to be
limited to the UFD system, but to reside in a more general step(s)
in protein degradation shared by these two model substrates.
| |
DISCUSSION |
Genetic evidence for separable functions of SRP1.
The
role of importin (karyopherin ) and its yeast homolog Srp1p in
NLS-dependent nuclear protein import has been well established. Genetic
experiments described in this article demonstrate that Srp1p carries
out at least one additional important function, unrelated to its NLS
receptor function. The first evidence for this conclusion is the
intragenic complementation of the srp1-31 ts mutation and
the srp1-49 ts mutation. It should be noted that intragenic
complementation could be observed for a protein which functions as a
homodimer consisting of two identical subunits; one mutational
alteration at a site disrupting the functional conformation of the
dimer could conceivably be suppressed by another "compensatory"
amino acid alteration at a different site of the same protein
molecule. However, the NLS receptor function of Srp1p is carried out as
a monomer (or as a heterodimer complexed with Kap95). It was
demonstrated that, in the absence of NLS peptides, purified
Xenopus importin forms aggregates at higher
concentrations, but that importin with a bound SV40-type NLS
peptide remains a monomer (32). Crystal structure analysis
of a 50-kDa fragment of Srp1p with and without an NLS peptide also
supports this conclusion (5). Thus, intragenic
complementation of srp1-31 and srp1-49 cannot be
explained by the model of formation of a functional homodimer with NLS
receptor function. Instead, this complementation demonstrates that the
function affected by the srp1-49 mutation is separate from
the NLS receptor function affected by the srp1-31 mutation.
This intragenic complementation also shows that the mutational defect
caused by srp1-31 is specific rather than a nonspecific heat
inactivation of the protein; the mutant protein Srp1-31p is defective
in the NLS protein transport function at nonpermissive temperatures but
is able to carry out another function that is affected by the
srp1-49 mutation at the same nonpermissive temperatures. We
also note that both Srp1-31 and Srp1-49 mutant proteins are stable at
38°C for at least 9 h (unpublished experiments; 48a). This observation excludes the (unlikely)
possibility that the observed intragenic complementation is a result of
stabilization of a mutant protein(s) by some kind of interaction
between the two proteins.
The second evidence is our finding that a high dosage of either
STS1 or RPN11 suppresses srp1-49 but
not srp1-31. This suppression in srp1-49
cells is correlated with suppression of decreased
ubiquitin-dependent protein degradation, as assayed by the use of a
model substrate, Ub-P-gal. No such stabilization of
Ub-P-gal degradation was observed for srp1-31. It would
be difficult to explain defects in Ub-P-gal degradation observed in
srp1-49 as a consequence of a defect in the NLS
receptor-nuclear protein import function, as further discussed below.
The amino acid residue serine 116 affected by
srp1-31 (S116F) (55) is an invariant
residue among all published Srp1p homologs from a variety of organisms.
The S116F mutation at this residue likely disrupts the structural
integrity of the NLS binding site, as discussed by Conti and coworkers
(5), based on the crystal structure of an Srp1p-NLS peptide
complex, and this is indeed consistent with the experimental
observations (44). The amino acid residue glutamic
acid 145 altered by the srp1-49 mutation (E145K)
(55) is located on the opposite, convex face of the Srp1p structure, away from the concave face where the known NLS binding
sites are located. This is also consistent with our observations that
mutation srp1-49 shows no defects in NLS binding or in
the import of any protein substrate tested so far at nonpermissive temperatures (44; our unpublished
experiments). As mentioned above, in vivo complementation of
srp1-31 with srp1-49 implies the absence of any
significant effect of this mutational alteration on the NLS receptor
function of Srp1p.
One possible model to explain our observations is that the
srp1-49 mutation is specifically defective in the transport
of some proteasome components but is not defective in the transport of
many other proteins, whereas the srp1-31 mutation is
opposite, i.e., is defective in the transport of many NLS-containing
proteins but not in the transport of the proteasome components.
According to this model, Srp1p functions in protein transport using two entirely different mechanisms: one is the well-established mechanisms, i.e., by binding the classical SV40-nucleoplasmin-type NLS peptides at
the concave face of the Srp1p protein structure; and the second is an
ad hoc and undefined mechanism without using the classical NLS-Srp1p
interaction. This second mechanism is not affected by the
srp1-31 mutation but is somehow affected by the
srp1-49 mutation; that is, this mechanism assumes binding of
cargo proteins perhaps to the convex face of the protein rather than to
the well-established NLS-interacting concave face. We originally
considered the possibility that high-dosage suppression of
srp1-49 by STS1 or RPN11 might reflect
a possible failure of transport of Sts1p or Rpn11p or related proteins
to the nucleus catalyzed by Srp1p. However, no defect in Sts1p
transport to the nucleus was observed in srp1-49 mutant
cells, as described in this paper, and no interaction of Srp1p with
Rpn11p was detected in the two-hybrid system or in vitro. Thus, while
we cannot exclude the above-mentioned model completely, we think that
it is probably unlikely.
Ubiquitin-dependent protein degradation is affected by the
srp1-49 mutation.
An important clue to the nature of
the Srp1p function affected by the srp1-49 mutation came
from the discovery of RPN11 as a high-dosage suppressor of
the srp1-49 mutation. RPN11 is a component of the
19S regulatory particle of the proteasome and shows 65% identity to
its human counterpart Poh1 (8, 47). Rpn11p contains a highly
conserved sequence with similarity to the active-site Cys box seen in
many deubiquitinating enzymes, and its possible function within the
proteasome was speculated on in this connection (8).
However, deubiquitination activity of isolated Rpn11p itself has not
been demonstrated, and the significance of this similarity is
not clear.
The Schizosaccharomyces pombe homolog of RPN11,
pad1+, was originally isolated as a gene which
confers resistance to staurosporine and other drugs when expressed in
high dosage (43). The increased resistance to the drugs was
explained by suggesting that overexpression of RPN11 caused
stabilization of unstable transcription factors, such as Pap1, required
for the expression of genes responsible for the drug resistance
(31, 47). However, no experimental tests have been carried
out to examine the validity of this explanation. Using the model
substrate Ub-P-gal for the ubiquitin-proteasome system, we have
found that this model substrate is significantly more stable in the
srp1-49 cells than in wild-type cells or the srp1-31 mutant cells. Overexpression of RPN11 in
srp1-49 cells decreased the stability of this model
substrate close to the wild-type level, although no such effects, i.e.,
further decrease in the stability, were observed in the control
wild-type cells (Fig. 7 and unpublished experiments). It
appears that the srp1-49 mutation causes some defects,
directly or indirectly, in the activity of the ubiquitin-proteasome
system and that overexpression of RPN11 somehow restores its
activity. A similar mutational defect, increased stability of protein,
was also observed for another model substrate, Leu-gal. It is known
that initial steps of degradation of Ub-P-gal are through the UFD
pathway, whereas those of Leu-gal degradation take place
through the N-end rule pathway (13, 14, 16). These two
pathways do not share ubiquitination enzymes E2 or E3, but share later
steps involving proteasome activity. Thus, the defect caused by the
srp1-49 mutation might be in a step(s) involving the
proteasome. However, mutations in proteasome subunits were often
reported to cause accumulation of polyubiquitinated protein substrates.
No increased accumulation of polyubiquitinated forms of Ub-Pro-gal
or Leu-gal in the srp1-49 mutant was observed in our
experiments. Therefore, it is possible that a
ubiquitination-polyubiquitination step(s) of these model substrates is
somehow affected by the mutation regardless of the kinds of E2-E3
ubiquitination enzyme systems.
Possible significance of an Srp1p-Sts1p complex in
ubiquitin-dependent protein degradation.
The function of Srp1p
affected by srp1-49, i.e., the function related to protein
degradation, as discussed above, may be carried out by a complex
containing Srp1p and Sts1p, and a direct interaction of these two
proteins may be important for this function. First, Srp1p and Sts1p
interact in vivo, as revealed in the two-hybrid system, and interact
directly in vitro. Second, a protein complex containing these two
proteins exists in cell extracts, as shown by coimmunoprecipitation
experiments. Third, the ts phenotype of srp1-49 can be
suppressed by a high dosage of STS1, and this suppression is
abolished by an sts1 mutation that disrupts the Srp1p-Sts1p
interaction detected by the two-hybrid system. Fourth, the same
sts1 mutation, which does not cause growth defects by itself, causes a synthetic growth defect when combined with
srp1-49, i.e., a more severe ts phenotype. In addition,
RPN11 and STS1 interact in the two-hybrid system.
Therefore, the main function of STS1 may also be related to
protein degradation. In fact, we observed that overexpression of
STS1 restores the decay rate of Ub-P-gal in
srp1-49 cells close to the wild-type level, and this may be
the basis of suppression of the srp1-49 mutation by a high dosage of STS1. Several previous observations can be
explained on the basis of the proposed new function (related to protein degradation) for both STS1 and SRP1, as discussed below.
First, SRP1 was originally identified as a suppressor of
certain ts mutations in the A190 subunit of Pol I (54).
Several point mutations of SRP1, such as SRP1-1
and SRP1-2, were isolated as suppressors. These mutations,
when separated from the rpa190 mutations, did not show any
phenotype by themselves, but the suppressor activity was dominant over
the wild-type SRP1 (54, 55). The rpa190 ts mutations, suppressed by these SRP1
suppressors, such as rpa190-1 and rpa190-5,
caused a large decrease in the amount of A190 at nonpermissive
temperatures, apparently due to an instability of mutant A190 protein
(26). One possibility that we considered based on the NLS
receptor function of Srp1p was an increase in the efficiency of binding
of A190 (or a complex containing A190, e.g., Pol I) to Srp1p, leading
to an increased supply of A190 (or a Pol I precursor or Pol I) to the
nucleus, counteracting the instability of the mutant protein. We tested
this possibility by constructing a CEN plasmid carrying the
srp1 gene (srp1-2,31) containing both the
SRP1-2 suppressor mutation and the srp1-31 mutation. We found that the Srp1p carrying these two mutations was more
severely ts in NLS binding in vitro than that carrying the
srp1-31 mutation only, and yet the plasmid introduced into the rpa190-1 mutant suppressed the ts phenotype (our
unpublished experiments). Thus, the suppressor activity was not
abolished by inactivation of the NLS binding-NLS protein transport
function. Therefore, we suggest that the suppressor mutations such as
SRP1-2 affect another function of Srp1p which is related to
protein degradation and counteract the degradation of the unstable
mutant Pol I subunit protein.
A similar interpretation can be applied to the reported suppression of
the rna15-2 mutation by a mutation in STS1
(1). Rna15p is an essential component of cleavage factor I
that, together with Rna14p, is involved in cleavage and polyadenylation
of mRNAs (27). For rna15-2, the observed defect
in 3'-end mRNA processing was due to unstable Rna15-2 protein at high
temperature. A mutation in STS1 partially corrects this
protein instability and so partially rescues 3'-end mRNA processing
(1). Since Srp1p and Sts1p appear to carry out a function as
a complex, as shown in this paper, we suggest that this suppressor
mutation in STS1 may involve stabilization of the unstable
nuclear protein Rna15-2p. Increased amounts of the protein rescue the
defect of rna15-2 at high temperature.
Liang et al. (22) originally discovered that a high dosage
of STS1 suppresses the ts phenotype of sec23-11
but not the ts phenotype of sec23-1. These authors reported
that a shift to the nonpermissive temperature leads to accumulation of
the ER form of a secreted protein, invertase. Overexpression of
STS1 alleviated this accumulation of invertase, which led
them to suggest that STS1 corrects the secretion defects of
this mutant. However, they have not followed the fate of the ER form of
invertase, and so it is not clear if overexpression of STS1
leads to transport of the ER form of invertase to the cell surface. The
ubiquitin-proteasome system is also involved in the degradation of
mutant secretory proteins that are accumulated in the ER (reviewed in
reference 34). It is therefore likely that a high
dosage of STS1 stimulates the degradation of proteins
accumulated in the ER, in the same way as it stimulates degradation of
Ub-P-gal in srp1-49 cells, leading to suppression of the
ts phenotype of sec23-11. In fact, we found that like
STS1, RPN11 can also suppress the ts defect of
sec23-11 (our unpublished experiments) (48a),
which supports our interpretation of the results published by Liang et
al. (22).
Role of SRP1 and STS1 in mitosis.
Although the function of Srp1p as the NLS receptor in protein import
has been well established, we have argued that Srp1p may also carry out
an additional essential function(s) concerned with the regulation of
protein degradation. It has been well established that regulated
protein degradation through the ubiquitin-proteasome system is
essential for progression of the cell cycle (reviewed in references
33 and 50). Mutations in
proteasome components or in the anaphase-promoting complex (a ubiquitin
protein ligase E3) are known to cause mitotic arrest. In fact, some
mutations in RPN11, which encodes a proteasome regulatory
subunit and is a high-dosage suppressor of srp1-49, have
recently been shown to cause a mitotic arrest (38;
see reference 31 for mutations in the S. pombe homolog pad1+). The basis of mitotic
arrest observed in these instances were shown or interpreted to be due
to mutational defects in the regulated proteolysis of certain key
proteins, such as mitotic cyclins and Pds1p (Cut2), which were shown to
be essential for progression of mitosis (33, 50, 52). Thus,
the mitotic arrest phenotype evident in the srp1-49 mutant
at nonpermissive temperatures (55) or after depletion of
Srp1p (20) can now be explained on the basis of defects in
protein degradation, as observed in the present work using model
substrates for the ubiquitin-proteasome system. However, we have not
determined the identity of the natural protein substrates whose
stability is affected by the srp1-49 mutation. In this
connection, we note that the srp1-31 mutation was reported to cause a clean mitotic arrest at nonpermissive temperatures and
degradation of Clb2p is defective under these conditions
(24). This observation was interpreted on the basis of
defects in nuclear import of some cell cycle regulators
(24). In our hands, however, mitotic arrests were clearly
evident for srp1-49 but not for srp1-31 (our
unpublished experiments). The reason for this discrepancy is not clear.
As already discussed, we favor our interpretation that the mitotic
arrest observed for srp1-49 is a consequence of defects in
the function related to protein degradation and not a consequence of
defects in nuclear import of hypothetical cell cycle regulatory proteins.
STS1, which was also called DBF8, was
independently identified as a gene essential for nuclear division and
segregation. Strains carrying dbf8 mutations show a mitotic
arrest phenotype at nonpermissive temperatures similar to that seen for
srp1-49 (12). This phenotype is consistent with
our conclusion that Sts1p, like Rpn11p, plays a role in the regulation
of protein degradation through the ubiquitin-proteasome system.
Another observation relevant to our conclusion on the new function of
Srp1p separate from its NLS receptor function is a recent work by
Matsusaka et al. (25) on the identification of S. pombe gene cut15+ as a homolog of
SRP1. These investigators found that a ts mutation in
cut15+ shows a mitotic arrest at nonpermissive
temperatures, presumably due to defects in chromosome condensation.
Surprisingly, the mutation caused a defect in NLS binding activity of
the protein in vitro, and yet no defects in protein import were
observed in vivo. They concluded that cut15+, a
homolog of SRP1 is essential for mitotic chromosome
condensation, but its role in nuclear protein import is dispensable
(25). Their results support our conclusion that Srp1p has a
function which is separable from the NLS receptor function. We note
that chromosome condensation appears to be defective in
srp1-49 mutants at nonpermissive temperatures
(55) and in Srp1p-depleted cells (20, 55). It
will be interesting to examine whether there is any defect in
activities of the ubiquitin-proteasome system in the cut15
mutant analyzed.
We also note that a ts mutation (cse1-2) in CSE1,
which encodes the yeast homolog of mammalian CAS, shows a mitotic
arrest phenotype at nonpermissive temperatures which is very similar to
srp1-49 (55), including a significant percentage
of arrested cells with spindle orientation defects (41).
Both yeast Cse1p and mammalian CAS have been demonstrated to function
in recycling Srp1p back to the cytoplasm after completion of nuclear
protein import (11, 18, 46). Consequently, the phenotypes of
cse1 mutations and various genetic interactions between
CSE1 and SRP1 have been explained solely on the
basis of the functions of these two genes in nuclear protein transport.
Schroeder et al. (41) reported suppression of
srp1-49 but not srp1-31 by a high dosage of
CSE1. Since srp1-49 does not show any defect in
nuclear protein import, this observed suppression is not easy to
explain on the basis of the Srp1p-recycling function of Cse1p. It is
known that the CAS protein is associated with microtubules, including
mitotic spindles (40), and its depletion by the use of
antisense RNA leads to mitotic arrest in mammalian cells
(29). The S. pombe Srp1p homolog,
cut15 protein, was also shown to be clearly associated with
the mitotic spindle, in addition to other intranuclear sites, during
late mitosis (25). It is known that degradation of a microtubule-associated protein, Ase1p, is mediated by the
anaphase-promoting complex-proteasome system in S. cerevisiae and that inappropriate expression of nondegradable
Ase1p during G1 caused mitotic arrest (15).
Thus, it is possible that Cse1p, like Srp1p, has functions important
for mitosis that are independent of its role in nuclear transport and
that suppression of defects in srp1-49 cells by a high
dosage of CSE1 is related to the proposed function of
regulation of protein degradation.
The proposed role of Srp1p in mitosis is also consistent with earlier
observations that pendulin, one of the Drosophila homologs of Srp1p, rapidly translocates from the cytoplasm to the nucleus at the
transition between G2 and M phase and is found associated with condensed metaphase chromosomes (19, 49). Such
observations suggest that an important function of pendulin is related
to mitosis. Since there are several distinct Srp1p homologs in higher
eukaryotes (17 and references therein), there might
be a functional differentiation among these homologs. In the yeast
S. cerevisiae, only a single protein species, Srp1p, exists
as an importin homolog. Thus, one can speculate that the yeast
Srp1p participates in a variety of nuclear functions through regulation
of degradation of various regulatory proteins and that these regulatory
functions might be divided among different Srp1p homologs in higher
eukaryotes. One can also speculate that during an earlier time in
evolution, Srp1p was a nuclear protein interacting with several other
nuclear proteins, such as Sts1p, and carrying out a specific
function(s) such as those discussed above, and that one of the importin
family members, that corresponding to the present-day Kap95p, was
the import machinery for Srp1p. Therefore, the protein import function
of Srp1p as a piggyback receptor might be a result of more recent
evolution and be limited to only certain kinds of nuclear proteins.
This speculative notion is supported by the fact that several importin
family members are engaged in nuclear import of a variety of
proteins without using Srp1p (for review, see references
9, 28, and
30).
In conclusion, the work described in this paper strongly indicates that
Srp1p carries out a function, almost certainly as a complex with Sts1p,
separate from its function as the NLS receptor in nuclear protein
import, and that this function is related to regulation of protein
degradation through the ubiquitin-proteasome system. Elucidation of the
molecular details of the proposed function is a subject for future studies.
We thank M. Waterman for helpful discussions and critical advice
on the manuscript. We thank J. Keener and K. Sutton in this laboratory
for suggesting the complementation experiments and for excellent
technical assistance, respectively. We thank J. Loeb for help in
construction of mutant strains and are grateful to K. Madura, P. Silver, F. Kepes, and A. Goldberg for providing plasmids and
strains. We also thank C. Carmen for help in preparation of the manuscript.
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(Karyopherin 
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Abstract
Introduction
Present address: Department of Developmental and Cell Biology,
University of California, Irvine, Irvine, CA 92697-2300.
