From the
Targeting of import substrate to nuclear pore complexes of
permeabilized vertebrate cells was previously shown to require a
protein complex composed of two subunits, termed karyopherin. Yeast
contain a homologue of karyopherin
Several factors that mediate import of proteins into nuclei of
permeabilized mammalian cells have been isolated and characterized. A
protein complex composed of two subunits termed karyopherin
In the yeast Saccharomyces
cerevisiae, protein import into nuclei was examined in vitro using spheroplasts that were permeabilized by
freeze-thaw(17) . In contrast to the mammalian import reaction,
docking of import substrate to the yeast nuclear envelope did not
require addition of cytosolic proteins(17) . To explain this
difference, it was proposed that previously identified NLS receptors of
yeast (none of them karyopherin homologues) remain tightly bound to the
NPCs of freeze-thawed cells and do not need to be added
back(17) . The recent identification of karyopherin in
vertebrates and the fact that karyopherin
Here
we report that yeast Srp1p is indeed the functional homologue of
vertebrate karyopherin
To facilitate isolation of yeast Srp1p/Kap60p and to
determine whether it, like vertebrate karyopherin
The data presented here demonstrate that yeast Srp1p is the
functional homologue of vertebrate karyopherin
Two lines of evidence suggest that yeast
karyopherin functions as an
We believe
that Kap60p functions like vertebrate karyopherin
Why was Srp1p/Kap60p detected as
a genetic suppressor of mutations in a subunit of RNA polymerase
I(19) ? Although at present there is no obvious answer, it is
likely that Kap60p performs a critical step in the assembly of RNA
polymerase I by mediating nuclear import of polymerase subunits.
We thank Drs. R. Erdmann, E. Johnson, U. Nehrbass, A.
Radu, and N. Schülke for help and advice. We thank J.
Fernández of the Rockefeller University Biopolymer Facility for
obtaining protein sequence for Kap95p, the Nomura Laboratory for
providing anti-Srp1/Kap60p antibodies, and D. Schnell for providing the
pET8c-pS/protA plasmid.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
named Srp1p, which was
initially identified as a genetic suppressor of mutations in a subunit
of RNA polymerase I. To determine whether yeast contain a karyopherin
complex that includes Srp1p as the karyopherin
homologue, we
genetically replaced Srp1p with a Srp1-Protein A chimera. Cytosol from
this strain contained a complex, composed of the chimera and a protein
of 95 kDa, that was purified using affinity chromatography on IgG
Sepharose. Microsequence analysis showed that the 95-kDa protein was
identical with a yeast protein encoded by gene L8300.15 on chromosome
XII. Sequence comparison revealed that the L8300.15 gene product is the
closest structural homologue of vertebrate karyopherin
. The yeast
and
karyopherin subunits were expressed in Escherichia
coli and were purified. When combined, they formed a heterodimeric
complex and were active in targeting import substrate to nuclear
envelopes of mammalian cells. We propose that all karyopherins function
as
/
heterodimers.
and
was shown to target substrates that contain a nuclear
localization sequence (NLS)
(
)to nuclear pore
complexes
(NPCs)(1, 2, 3, 4, 5) . A more
detailed analysis showed that karyopherin
mediates recognition of
the NLS substrate(2, 4, 6) , whereas karyopherin
functions to dock the karyopherin
/NLS-substrate complex to
a subgroup of nucleoporins that contain peptide
repeats(3, 7, 8) . Subsequent transport into the
nucleus is mediated by the small GTPase Ran (9, 10) and
a Ran interacting protein(11) . The mechanics of transport and
the involvement of other proteins such as Ran-GTPase activating
protein(s) (12), Ran-GDP/GTP exchange protein(s)(13) , and the
heat-shock cognate protein hsc70 (14, 15) remain to be
elucidated(16) .
is homologous to yeast
Srp1p (4, 18, 19, 20) suggested that
targeting of NLS substrates to NPCs in yeast is mediated by a homologue
of vertebrate karyopherin and that Srp1p is the
subunit.
and that it can be isolated from yeast
cytosol in a heterodimeric complex with a protein of 95 kDa that is a
functional homologue of vertebrate karyopherin
. Yeast karyopherin
and
were expressed separately in Escherichia coli,
and each was purified to homogeneity. Recombinant
and
assembled into a heterodimer in vitro and were required in
combination for NLS substrate recognition and docking to nuclear
envelopes in digitonin-permeabilized mammalian cells. The yeast
proteins are therefore termed Kap60p for karyopherin
subunit of
60 kDa and Kap95p for karyopherin
subunit of 95 kDa. The
alternate term Kap60p for Srp1p was deemed necessary to avoid further
confusion with the previously issued acronym SRP (signal recognition
particle) (21).
Strains
S. cerevisiae strains used were
DF5 (MATa/MAT trp1-1/trp1-1
ura3-52/ura3-52 his3-
200/his3-
200 leu2-3, 112/leu2-3, 112
lys2-801/lys2-801 gal/gal) (22) and the DF5-derived strains
CEY1 (Mata/Mat
KAP60/kap60
::URA3),
CEY1A (Mata KAP60), and CEY1B (Mat
kap60
::URA3, pRS315-Kap60-ProtA). Standard molecular biological and yeast
genetic techniques were used(23) . To construct CEY1,
nucleotides -450 to 1950 that contain the SRP1/KAP60 open reading frame (ORF) (19) were amplified from S.
cerevisiae genomic DNA by PCR using the primers 5`-ATT GAT CCC TCG
AGG TTA ACT TAA TCG ACC G-3` and 5`-CTA GGA AGA TCT TTC AGC TGT GGA-3`.
The PCR product was digested with XhoI and BglII and
inserted into XhoI-BamHI-digested pBluescript
(Stratagene). The 1.1-kilobase HindIII-EcoRV fragment
(nucleotides 149-1255) within the SRP1/KAP60 ORF was
replaced by the 1.1-kilobase HindIII-SmaI fragment
that contains the URA3 gene from vector pJJ244(24) .
The kap60
::URA3 deletion allele was transformed as a PvuII fragment into the diploid strain DF5. The replacement of
one chromosomal KAP60 copy by the deletion allele in the
Ura
transformant CEY1 was confirmed by Southern blot
hybridization. To express Kap60-ProtA in yeast, nucleotides -450
to 1626 that contain the SRP1/KAP60 ORF (19) were amplified by PCR from yeast genomic DNA using primers
that incorporate a XhoI site at the 5` end and an in-frame BamHI site replacing the stop codon of SRP1/KAP60.
The PCR fragment was inserted into XhoI-BamHI-digested CEN6-ARS-LEU2 vector
pRS315 (25) to generate pRS315-Kap60. The segment of the
staphylococcal Protein A gene (nucleotides 272-1104) that encodes the
five IgG binding domains was amplified from pET8c-pS/protA (26) by PCR using primers that incorporate an in-frame BamHI site at the 5` end and a stop codon followed by a BamHI site at the 3` end. The PCR product was digested with BamHI and ligated in pRS315-Kap60 to generate
pRS315-Kap60-ProtA, which was used to transform CEY1. Leu
transformants were selected and sporulated. After tetrad
dissection Ura
Leu
spores were
selected, one of them was named CEY1B. To verify the replacement of the
wild-type copy of SRP1/KAP60 by the plasmid-born SRP1/KAP60-ProtA, cell lysates of CEY1B were analyzed by
SDS-PAGE and ECL Western blotting (Amersham).
Purification of the Kap60-ProtA/Kap95p
Complex
Yeast strain CEY1B was grown in 5 liters of YPD medium
at 30 °C for 16 h to early stationary phase. Cells (80 g) were
harvested and converted into spheroplasts as described(27) ,
using only Zymolyase 20T. Spheroplasts were lysed in 300 ml of 50
mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF
with a Dounce homogenizer. Cell debris was removed by centrifugation at
12,000 g for 15 min at 4 °C, and the supernatant
was clarified by centrifugation at 200,000
g for 15
min at 4 °C. The high speed supernatant (25 mg of protein/ml) was
mixed with 3 ml of packed IgG-Sepharose beads (Pharmacia Biotech Inc.)
that were equilibrated in loading buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM PMSF), and the
mixture was incubated for 2 h at 4 °C in batch. Beads were
transferred to a column and washed with 30 bed volumes of loading
buffer and 2 bed volumes of 5 mM NH
OAc, pH 5.0.
Bound proteins were eluted with 10 bed volumes of 0.1 M
glycine-HCl, pH 3.0, and neutralized with 1 M Hepes-KOH, pH
7.4. The eluted 90-kDa protein was processed for microsequence analysis
as described(28) .
Purification of Recombinant Kap60 and Kap90 Proteins from
E. coli
KAP60 and KAP95 genes were amplified
separately from yeast genomic DNA by PCR using synthetic
oligonucleotides (5`-CCG GGA TCC ATG GAT AAT GGT ACA GAT TCT TCC ACG
AGC A-3` and 5`-CCG GGA TCC TTA ATT GAA TTG TTG GTT GAC ATT AGA ACC
A-3` for KAP60, and 5`-CCG GGA TCC ATG TCC ACC GCT GAA TTT GCT
CAA and 5`-CCG GGA TCC TTA TAA GGA TAA TTG ACG CTT CTG TTG-3` for KAP95) that incorporate a BamHI endonuclease
restriction site in-frame with the initiation codon and another after
the stop codon of each gene. The purified BamHI fragments were
ligated into vector pGEX-2TK (Pharmacia Biotech Inc.) to create
in-frame fusions with the glutathione S-transferase gene (GST). Plasmids that contained GST-KAP60 and GST-KAP90 gene fusions were introduced into E. coli strain BLR (Novagen). Fusion proteins were purified as follows.
Reagents used were obtained from Sigma, unless otherwise indicated.
Cells were grown in 1 liter of 2 YTA (Difco Laboratories) at 37
°C to a cell density of 1-2 A
units.
Cells were shifted to 28 °C, and
isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.5 mM to induce synthesis of fusion
proteins. After 1 h, cells were harvested at 4 °C by
centrifugation, and cell pellets were resuspended with 18 ml of chilled
lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl).
PMSF and lysozyme were added to final concentrations of 0.5 mM and 1 mg/ml, respectively, and the cell suspension was incubated
for 15 min at room temperature. Sodium deoxycholate and DNase I were
added to final concentrations of 1 mg/ml and 10 µg/ml,
respectively, and lysates were incubated again at room temperature for
15 min. Cell debris was removed by centrifugation at 20,000
g for 10 min at 4 °C, and the supernatant was filtered
through a 0.45-µm syringe filter (Schleicher and Schuell). The
filtrate was mixed with 0.5 ml of packed glutathione-agarose beads that
were equilibrated in transport buffer (TB: 20 mM Hepes-KOH, pH
7.4, 110 mM KOAc, 2 mM MgOAc, 1 mM EGTA, 2
mM dithiothreitol), and the mixture was incubated at 4 °C
for 1-6 h. Beads were collected by centrifugation at 2,000
g for 2 min at 4 °C and were washed 4 times with
15 ml of TB by repeated resuspension and centrifugation. To elute
fusion protein, beads were resuspended in 1 ml of TB with 10 mM reduced glutathione and were incubated for 10 min at 4 °C.
Pooled eluates (3 ml) contained fusion protein at an average
concentration of 0.5 mg/ml. To cleave Kap proteins from GST, thrombin
was added to eluates that had been concentrated (1.5 NIH units of
thrombin per 100 µg of GST-Kap60p and 3 NIH units per 100 µg of
GST-Kap95p), and the mixture was incubated for 10-30 min at room
temperature. Samples were chilled, and PMSF was added to a final
concentration of 0.5 mM. Proteins in the samples were resolved
in a Superdex 200 FPLC sizing column (Pharmacia Biotech Inc.) to yield
near-pure Kap proteins in fractions depleted of GST, thrombin, and
uncleaved fusion protein.
Nuclear Docking Assay
Assays were performed as
described previously (4, 29) with a few technical
variations that are outlined below. Buffalo rat liver cells were
permeabilized in 50 µg/ml digitonin in TB, during a 5-min
incubation at room temperature. Cells were then placed on ice and
washed twice with chilled TB. Reactions were incubated for 15 min on
ice and contained additions as indicated in the figure legends.
Completed reactions were washed twice with chilled TB, and cells were
fixed with 3% formaldehyde in TB. Cells were visualized and
photographed under a Zeiss Axiophot microscope (Carl Zeiss, Inc.).
, is associated
with a
subunit, we genetically replaced cellular Kap60p with a
Kap60p-Protein A chimera that could be easily isolated using
IgG-Sepharose chromatography. Immunoblot analysis of wild-type and
mutant cell lysates using anti-Srp1p(Kap60p) antibodies (19) showed that Kap60p was absent in mutant cells (Fig. 1A, compare lanes 1 and 2) but
was replaced by the Kap60p-Protein A chimera (lane 2). When
the same blot was probed with anti-rabbit IgG-horseradish peroxidase,
the chimera (lane 4) but not wild-type Kap60p (lane
3) was detected.
Figure 1:
Kap60-ProtA can
be isolated from yeast cytosol in a complex with Kap95p. A,
expression of Kap60-ProtA in yeast. Whole cell lysates (50 µg of
protein per lane) from yeast strains CEY1A (KAP60) (lanes
1 and 3) and CEY1B (kap60::URA3, pRS315-Kap60-ProtA) (lanes 2 and 4) were
subjected to 10% SDS-PAGE and analyzed by immunoblotting using
anti-Srp1p (Kap60p) antibodies (19) and ProtA-horseradish peroxidase
conjugates (lanes 1 and 2) or anti-rabbit
IgG-horseradish peroxidase conjugates (lanes 3 and 4); horseradish peroxidase conjugates were obtained from
Amersham. B, co-purification of Kap60-ProtA and Kap95 proteins
by IgG-Sepharose affinity chromatography. Fractions were analyzed by
10% SDS-PAGE and Coomassie Blue staining: lane 1, high-speed
supernatant of a cell extract of CEY1B (50 µg of protein); lane
2, flow-through (50 µg of protein); lane 3, combined
wash fractions; lane 4, concentrated acid eluate (3 µg of
protein).
The tagging of Kap60p with Protein A allowed
its purification from a high-speed supernatant of a cell lysate using
IgG Sepharose chromatography (Fig. 1B). Acid elution of
tightly bound proteins and subsequent analysis of the eluate by
SDS-PAGE showed two major bands in approximately stoichiometric amounts (lane 4). The slower moving polypeptide was the Kap60p-Protein
A chimera, as determined by immunoblot analysis (data not shown). To
determine the chemical nature of the faster moving protein, we
subjected it to microsequence analysis and obtained a peptide
(KQFYGQDWVIDYKRTRSGQLFSQATKD) that was identical with the C terminus of
a 95-kDa protein of unknown function, encoded by the essential gene L8300.15 on yeast chromosome XII (GenBank
accession number U19028). A protein homology search in GenBank
identified rat karyopherin
as the closest structural homologue of
the L8300.15 gene product, with 33% identity and 63% similarity (Fig. 2). Because of the high degree of similarity to vertebrate
karyopherin
and because of its apparent stoichiometric
association with yeast karyopherin
(Kap60p), we termed this
protein Kap95p. Taken together the data suggest that yeast, like
vertebrate cells, possess a cytosolic karyopherin complex.
Figure 2:
Dot-matrix comparison of yeast and rat
karyopherin sequences. Stringency: 10; window: 30 (Lasergene,
DNAstar). The amino acid sequences of yeast karyopherin
(Kap95p)
and rat karyopherin
are available in GenBank
,
accession numbers U19028 and L38644,
respectively.
To
determine whether yeast karyopherin functions in targeting import
substrate to NPCs, we expressed the two subunits separately in E.
coli and purified each to homogeneity. Molecular sieving of
isolated Kap60p and Kap95p showed that each migrated as expected for a
monomeric globular protein (Fig. 3, A and B).
When the two subunits were incubated together at 0 °C and the
mixture was subjected to molecular sieving, approximately 50% of the
subunits assembled into a heterodimeric complex (Fig. 3C). To test whether recombinant yeast karyopherin
subunits are competent to target an import substrate to NPCs, we
employed a well characterized assay that uses digitonin-permeabilized
mammalian cells(29) . Indeed, as previously shown for vertebrate
karyopherin subunits(1, 2, 4, 8) , both
yeast subunits were required to obtain docking of import substrate at
the nuclear envelope (Fig. 4, panel 3). Neither subunit
alone was sufficient to obtain docking (panels 1 and 2). Thus, we conclude that Kap60p and Kap95p are the
functional homologues of karyopherin in yeast.
Figure 3:
Recombinant Kap60 and Kap95 proteins
assemble into a 180-kDa heterodimer. A, 100 µg of isolated
GST-Kap95p were digested with thrombin and applied to a Superdex-200
FPLC sizing column, as described under ``Experimental
Procedures.'' Proteins in eluted fractions were resolved by
electrophoresis in a 9% polyacrylamide gel and were stained with
Coomassie Blue. B, 100 µg of isolated GST-Kap60p were
digested, fractionated, and analyzed as described in A. C, the digested products described in A and B were mixed and incubated for 30 min on ice, prior to fractionation
and analysis as described in A.
Figure 4:
The Kap60/Kap95 heterodimer promotes
docking of import substrates to nuclear envelopes. Nuclear docking
assays were performed using digitonin-permeabilized Buffalo rat liver
cells as described under ``Experimental Procedures.'' All
reaction mixtures contained 20 µg/ml fluorescent import substrate
(TRITC-labeled human serum albumin with conjugated peptides that
contain the NLS of SV40 large T-antigen (29), 1 mg/ml bovine serum
albumin, 1 mM ATP, 0.1 mM GTP, 5 mM phosphocreatine, 20 units/ml creatine phosphokinase, and protein
fractions as indicated below). Reactions contained 15 µg/ml (250
nM) purified Kap60p, 15 µg/ml (150 nM) purified
Kap95p, or both, as indicated. Cells were visualized and photographed
under a Zeiss Axiophot microscope. All panels correspond to reactions
that were incubated, photographed, and printed under identical
conditions.
and that it is
present in yeast cytosol in a heterodimeric complex with a novel 95-kDa
protein (Fig. 1). We show the latter to be the functional
homologue of vertebrate karyopherin
( Fig. 2and Fig. 4). We demonstrate that recombinant yeast karyopherin
(Kap60p) and karyopherin
(Kap95p) assemble into a heterodimeric
complex in vitro (Fig. 3) and are required in
combination to target import substrates to nuclear envelopes of
permeabilized mammalian cells (Fig. 4). Based on these data, we
propose that yeast karyopherin mediates the docking of import substrate
to nuclear envelopes of yeast. Proof awaits the development of a
karyopherin-dependent import assay in yeast (see introduction).
Vertebrates contain several homologues of karyopherin
(3, 4, 5, 6, 18) . Other
yeast homologues of karyopherin
have not been identified, but
there appear to be several homologues of karyopherin
.
(
)
/
heterodimer. First, neither
Kap60p nor Kap95p displayed docking activity when added alone (Fig. 4, panels 1 and 2); docking was only
detected when Kap proteins were mixed prior to a reaction (Fig. 4, panel 3), under conditions that allow the
formation of a 180-kDa heterodimer (Fig. 3C) (see
below). Second, Kap60 and Kap95 proteins were isolated from yeast
cytosol as a heterodimeric complex (Fig. 1). We concluded that
yeast karyopherin is a heterodimer based on the expected molecular size
of a 1:1 complex (65 + 100 = 165 kDa) (see Fig. 3)
and on the equimolar amount of Coomassie Blue stain adsorbed by each
subunit in the complex (Fig. 1B and 3C). Our
conclusion provides proof of our previous proposal that karyopherins
function as
/
heterodimers (3, 4, 8) and is fully consistent with the
results of others in that two distinct proteins are required to target
NLS substrates to NPCs(1, 2, 5) .
to recognize
the NLS substrate(4) , and Kap95p functions like vertebrate
karyopherin
(3) to dock the Kap60p/NLS-substrate complex
to nucleoporins that contain peptide
repeats(3, 7, 8) . We therefore suspect that the
reported genetic and physical interaction between Srp1p and the
nucleoporin Nup1p (30) is indirectly mediated by Kap95p; indeed,
the uncharacterized 95-kDa protein that co-purifies with Nup1 and Srp1
proteins (30) is probably Kap95p. Docking of yeast karyopherin
to multiple sites on the NPC would explain previous immunofluorescence
data that show co-localization of Srp1p and nucleoporins in a punctate
pattern on the nuclear surface(19) . We recently showed that
vertebrate karyopherin
enters the nucleus, whereas karyopherin
remains at the NPC(8) . If Kap60p functions in a similar
manner in yeast, it would explain the finding that Srp1p is also
localized in the nucleoplasm (19).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.