From the Department of Chemistry, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan and the
§ Department of Integrated Biosciences, Graduate School of
Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha,
Kashiwa 277-8562, Japan
Received for publication, October 25, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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During the mating of yeast Saccharomyces
cerevisiae, two haploid nuclei fuse to produce a diploid nucleus.
This process requires the functions of BiP/Kar2p, a member of the Hsp70
family in the endoplasmic reticulum, and its partner protein, Jem1p. To
investigate further the role of BiP and Jem1p in nuclear fusion, we
screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p. Nep98p is an essential integral membrane protein
of the nuclear envelope and is enriched in the spindle pole body (SPB),
the sole microtubule-organizing center in yeast. Temperature-sensitive
nep98 mutant cells contain abnormal SPBs lacking the
half-bridge, suggesting the essential role of Nep98p in the
organization of the normal SPB. Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating. Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent
karyogamy and in Jem1p-independent nuclear division.
In the sexual phase of yeast Saccharomyces cerevisiae,
haploid cells of opposite mating types mate to produce diploid cells. After cell fusion, the two haploid nuclei fuse to form a diploid nucleus (1). This process, karyogamy, can be dissected into two steps:
nuclear congression and nuclear fusion (1, 2). Analyses of yeast
mutants defective in nuclear fusion have revealed the involvement of
BiP/Kar2p, an Hsp70 molecular chaperone in the endoplasmic reticulum
(ER)1 (3). BiP also performs
functions including protein import into the ER and ER-related protein
degradation (4, 5). BiP has three functional partner proteins in the
DnaJ family: Scj1p, Sec63p, and Jem1p (6-8), and the functions of BiP
are specified by different DnaJ-like proteins. Protein import into the
ER lumen is mediated by BiP and Sec63p, and protein aggregation is
prevented in the ER lumen by BiP with Scj1p and Jem1p (9). Nuclear
fusion is facilitated by the Sec63p complex and Jem1p; the zygotes of kar2, sec63, and jem1 mutants contain
two closely opposed haploid nuclei that do not fuse (2, 8, 10).
As a step toward understanding the mechanism of nuclear membrane fusion
by BiP with Jem1p, we have screened for proteins that interact with
Jem1p using the yeast two-hybrid system. The identified protein,
Nep98p, is an essential integral membrane protein of the nuclear
envelope and is enriched in the spindle pole body (SPB), the sole
microtubule-organizing center of budding yeast and a functional
homologue of the centrosome in mammalian cells (11). The
temperature-sensitive (ts) nep98 mutant cells
have abnormal SPBs lacking the half-bridge and show defects in both mitotic nuclear division and karyogamy, suggesting that Nep98p is
essential for SPB organization and function.
Strains and Culturing Conditions--
Yeast strains SEY6210
(MAT Two-hybrid Screening--
A bait plasmid for two-hybrid
screening, pNY6, was constructed by cloning a 2.0-kb
ScaI/XhoI fragment of pSNJ2 (8) into the
SalI and SmaI sites of pGBD-C2 (13). Strain
PJ69-4A carrying plasmid pNY6 was transformed with the yeast two-hybrid
library. About 1 × 108 colonies were screened for the
His+ and Ade+ phenotype.
Plasmids and Strain Constructions--
The NEP98 gene
was cloned by PCR from the yeast genomic DNA using the primers
5'-GCGCTCGAGCCTTCAATTTTGGAG-3' and 5'-CGCGAGCTCTGGTGGTAATTCGAG-3'. The
amplified 3.2-kb fragment was digested with XhoI and
SacI and introduced into pRS316 (15) to generate pSNY3.
pSNY5 and pSNY6 were constructed by introducing a 3.2-kb
XhoI/SacI fragment of pSNY3 into pRS314 (15) and
pYO326 (16), respectively. pNTY5, a plasmid for the
NEP98-GFP fusion gene, was constructed by introducing the
3.1-kb fragment containing the entire NEP98 open reading
frame and its promoter. The fragment was PCR-amplified from
pSNY3 using the primers 5'-GCGGAGCTCCTTCAATTTTGGAG-3' and
5'-GCGCTCGAGTGATCTAGCTCATCTTG-3' and was digested with XhoI
and SacI into pAK13, which was constructed from pAK2 (17) by
replacing the GFP gene with the EGFP-myc fusion gene. The 3HA epitope tag, comprised of three tandem repeats of the
influenza hemagglutinin (HA) epitope (YPYDVPDYA), was introduced at the
N terminus of Nep98p at the DNA level by oligonucleotide-directed mutagenesis (18). A series of deletion mutants of the NEP98 gene were constructed by oligonucleotide-directed mutagenesis and
introduced into pRS314. pSNJ21 was constructed by introducing the
2.4-kb SacI/XhoI fragment of pSNJ2 into the
SacI and XhoI sites of pYO324 (16).
A 2.2-kb fragment containing the NEP98 gene was
PCR-amplified using primers 5'-GCGGCATGCATGAATAACTCAAATGAGC-3' and
5'-GTTCAAGGTTGGAATTCAG-3' and was introduced into the SphI
and EcoRI sites of pUC119 to yield pUCNEP98. A null allele
of NEP98, nep98::HIS3, was
constructed by replacing the 1.2-kb StuI/XbaI
fragment of pUCNEP98 with the 1.8-kb SmaI/XbaI
fragment of pJJ215 (19). The resulting plasmid, pUCDnep98, was digested
with SphI and EcoRI and introduced into a diploid
strain, SNY621D. His+ colonies were selected, and
disruption of the NEP98 gene was confirmed by PCR. The
NEP98/ Temperature-sensitive nep98 Mutants--
To screen ts
alleles of the NEP98 gene, pSNY5 mutagenized with
hydroxylamine (14) was introduced into SNY1061-2B. Trp+
colonies were replica-plated onto SCD ( Anti-Nep98p Antiserum--
A DNA fragment corresponding to
codons 238-682 of Nep98p was amplified by PCR and introduced into the
NdeI and XhoI sites of pET21a (Novagen). The
resulting plasmid, pET-NEP98, was introduced into BL21(DE3), and
expression of the C-terminal region of Nep98p was induced by adding 1 mM isopropyl- Microscopy--
Immunofluorescence microscopy was performed as
described previously with the anti- Miscellaneous--
The procedures were described previously for
the preparation of yeast microsomal fraction (21), the extraction and
trypsin digestion of membrane proteins (8), and the metabolic labeling of yeast cells and immunoprecipitation (20). For cross-linking experiments, spheroplasts labeled with 35S-amino acids were
broken open via the addition of 0.1% Triton X-100 and were incubated
with various concentrations of dithiobis(succinimidyl propionate)
(DSP) for 20 min on ice. The cross-linking was quenched through
the addition of 50 mM Tris-HCl, pH 8.0, and the reaction mixtures were subjected to immunoprecipitation.
Nep98p Is a Nuclear Envelope Protein Interacting with
Jem1p--
We performed yeast two-hybrid screening to look for
possible partner proteins for Jem1p in nuclear membrane fusion. A
fusion between Jem1p and the DNA binding domain of Gal4p was used as bait to screen 1 × 108 colonies, isolating 323 positive clones. Of the 270 positive clones sequenced, 50 clones were
found to contain a part of the open reading frame YJL019w of
unknown function. DNA sequencing of this region revealed insertion of
guanine downstream of G1851 in the YJL019w open
reading frame of the yeast genome data base, leading to a frameshift
and fusion with YJL018w to yield a new open reading frame
for 682 amino acid residues (Fig.
1A). We named the gene
NEP98 (the 98-kDa nuclear envelope
protein) on the basis of the properties of its gene product
described below. Tetrad analysis of the
NEP98/nep98::HIS3
heterozygous diploid showed that, among 21 tetrads dissected, all
produced two viable spores and that all viable spores showed the
His
A fusion protein consisting of Nep98p followed by GFP is fully
functional as Nep98p because it complemented the nep98-null mutant when expressed from a single-copy plasmid (data not shown). The
expressed Nep98p-GFP fusion protein exhibits perinuclear staining of
GFP fluorescence with one or two bright dots (Fig. 1B,
panels a and c). The anti-Nep98p antibodies
recognized a 98-kDa protein in the total cell extract, and indirect
immunofluorescence microscopy with the antibodies showed a similar
pattern of perinuclear staining (data not shown). Immunofluorescence
microscopy with the anti-
Nep98p possesses a possible membrane-spanning segment of residues
155-175 (Fig. 1A). We thus treated the yeast microsome
fraction with various reagents and analyzed proteins by immunoblotting with the anti-Nep98p antibodies after centrifugation (Fig.
2A). Nep98p was resistant to
extraction with 1 M NaCl, 2 M urea, or 0.1 M sodium carbonate, whereas soluble and peripheral membrane proteins such as BiP and Jem1p were extracted under the same conditions (data not shown), indicating that Nep98p is indeed an integral membrane
protein. Nep98p was not extracted with 1% Triton X-100 but was
extracted with 1% Triton X-100 plus 1 M NaCl, suggesting that protein-protein interactions are involved as well in the membrane
association of Nep98p. Because Nep98p was digested with externally
added trypsin to generate a 78-kDa fragment that was recognized by the
antibodies to the C-terminal region of Nep98p (
Physical interactions between Nep98p and Jem1p were
confirmed by cross-linking. Cells expressing Nep98p and Jem1p-3HA were converted to spheroplasts, labeled with 35S-amino acids,
lysed with 0.1% Triton X-100, and treated with a homobifunctional
cross-linker, DSP. The proteins subsequently were extracted and
subjected to the first round of immunoprecipitation with the
anti-Nep98p antibodies. The immunoprecipitates were treated with
dithiothreitol to cleave a disulfide bond of the cross-linker and were
subjected to the second round of immunoprecipitation with the
anti-Nep98p antibodies or the anti-HA monoclonal antibody. Jem1p-3HA
was precipitated with the anti-HA antibody only when cells were treated
with DSP (Fig. 2C), indicating that Nep98p indeed interacts
with Jem1p in yeast cells.
Nep98p Is Essential for Nuclear Division and Normal SPB
Functions--
To analyze the functions of Nep98p, we constructed
ts alleles of the plasmid-borne NEP98 gene. One
mutant allele, nep98-7, contained a mutation of
Asn597
We analyzed the nuclear morphology of the large budded cells by
4',6-diamidino-2-phenylindole (DAPI) staining.
We next analyzed the spindle morphology in nep98-7 cells at
restrictive temperatures. Four h after a shift from 23 to 37 °C, wild-type and nep98-7 cells were processed for
immunofluorescence microscopy using the anti-
The SPB spans both of the nuclear envelope membranes and is composed
generally of three major layers, an outer plaque on the cytoplasmic
side, the inner plaque on the nucleoplasmic side, and the central
plaque. The half-bridge is a specialized region of the nuclear envelope
and is continuous to one side of the SPB. Electron microscopy analyses
allowed us to examine in more detail the defects in the SPB
organization of nep98-7 cells. Enlarged SPBs with elaborated
inner plaques are observed in nep98-7 cells at both 23 and
37 °C (Fig. 4, C-F). We
analyzed the 24 SPBs in the nep98-7 cells by serial
sectioning and did not find the half-bridge in the mutant SPBs,
although we did observe the half-bridge in 16 of 25 SPBs in the
wild-type cells (Fig. 4A). The SPBs often were observed
apparently inside the nucleus in nep98-7 mutant cells (Fig.
4, C-F). The half-bridge and the outer plaque
function as the sites for cytoplasmic microtubule attachment, and the
inner plaque is the site for spindle microtubule attachment (11). The lack of the half-bridge and the elaboration of the inner
plaque in the nep98-7 SPBs likely affected the balance of
forces exerted by cytoplasmic and spindle microtubules and pulling on
the SPB. An imbalance in these forces led to an increase in the inward force, thereby making the SPB collapse in the nucleus. Fig.
4G shows an electron micrograph of a section of a large
budded nep98-7 cell with a dividing nucleus at 37 °C. In
contrast to wild-type cells, the SPB is localized around the bud neck
in nep98-7 cells, and the serial thin sections show that the
axis of the short spindle is formed perpendicular to the long axis of
the cell (Fig. 4, G-1-G-3). Nucleation of microtubules at
both ends of the spindle indicates the presence of the SPB at both
ends, suggesting that the defects of the mutant occurred after SPB
duplication.
Nep98p Has Functional Domains on Both Sides of the
Membrane--
To analyze the functional domain organization in Nep98p,
we constructed a series of partial deletion mutants of Nep98p as shown
in Fig. 5A. Nep98p takes the
Nout-Cin topology in the nuclear envelope
membrane, and immunofluorescence microscopy using the anti-HA-tagged
monoclonal antibody showed that all of the deletion mutants are
localized to the nuclear envelope as well (data not shown). Although
the deletion mutant lacking 32 C-terminal residues (
The cytoplasmic N-terminal part of Nep98p contains a cluster of acidic
residues. Cells expressing the deletion mutant lacking these residues
( Nep98p Functions in Nuclear Fusion during
Mating--
Because Nep98p was isolated as a Jem1p-interacting
protein, we tested whether Nep98p plays a role in nuclear fusion during mating. Wild-type or nep98-7 mutant cells of the opposite
mating types were mated at 23 °C, and the nuclei of the zygotes were stained with DAPI. Whereas >90% of the wild-type zygotes contained a
single nucleus (Kar+), 59% of the nep98-7
zygotes contained two or more nuclei that had failed to fuse; 37% had
nuclei close to each other (fusion In the present study, we have identified Nep98p as a partner
protein for Jem1p, a DnaJ-like protein of the yeast ER lumen. Nep98p is
a nuclear envelope protein that is enriched in the SPB. The ts
nep98 mutant strain exhibits defects in the SPB organization and
fails to achieve both nuclear division at restrictive temperatures and
nuclear fusion during mating, even at permissive temperatures. Because
the SPB has essential functions in nuclear division as well as nuclear
fusion, Nep98p likely mediates these processes through fabrication of
the SPB.
Jem1p facilitates the nuclear membrane fusion during mating
(8, 23), and the present results raise the possibility that Jem1p plays
a role in nuclear fusion through regulating the functions of Nep98p. It
is to be noted that deletion of the Jem1p-interacting domain of Nep98p
is lethal for yeast cell growth, whereas Jem1p is not essential for
yeast cell growth. Moreover, the jem1-null mutant does not
show defects in nuclear division (data not shown). In the nuclear
fusion process, two SPBs of haploid nuclei fuse to produce a single SPB
of a diploid nucleus. The SPB fusion is observed only during mating,
and this process appears to start from the satellite on the half-bridge
(24). Thus it is likely that Nep98p requires Jem1p and BiP only in the
SPB fusion process. Electron microscopy showed that the
The nep98-7 mutant showed defects in the nuclear congression
in addition to the nuclear fusion in karyogamy. The nuclear congression step is dependent on cytoplasmic microtubules, which nucleate from the
outer plaque of the SPB in vegetative cells (11, 24). Mating
pheromones induce the rearrangement of cytoplasmic microtubules at the
SPB, and the half-bridge functions as the site for cytoplasmic microtubules to organize into mating cells (24, 25). The nuclear congression defect in nep98-7 cells probably occurs because
of the aberrant SPB structure that lacks a visible half-bridge
structure. Nep98p thus is a multifunctional organizer of the SPB.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ura3 leu2 trp1 his3 lys2 suc2) and
SEY6211 (MATa ura3 leu2 trp1 his3 ade2
suc2) (12) were used as the parental strains for the mutants
constructed in this study. SEY621D was constructed by mating SEY6210
with SEY6211. PJ69-4A (MATa ura3 leu2 trp1 his3 gal4 gal80
GAL2-ADE2 LYS2::GAL1-HIS3
met2::GAL7-lacZ) (13) was used for the yeast two-hybrid assay. Yeast cells were grown in YPD (1% yeast extract, 2%
polypeptone, and 2% glucose) or in SD (0.67% yeast nitrogen base
without amino acids and 2% glucose) supplemented appropriately as
described previously (14). SCD medium is SD containing 0.5% casamino
acids. A sulfate-free synthetic minimal medium was used for metabolic
labeling of yeast cells. A quantitative mating assay was performed as
described previously (14).
nep98 heterozygous diploid was named SNY1054. SNY1061-1D (MATa
nep98::HIS3 ura3 leu2 trp1 his3 lys2
suc2 YCp[NEP98 URA3]), SNY1061-2B
(MATa nep98::HIS3 ura3
leu2 trp1 his3 suc2 YCp[NEP98 URA3]), and SNY1061-3C
(MAT
nep98::HIS3 ura3 leu2
trp1 his3 ade2 suc2 YCp[NEP98 URA3]) were constructed
by dissecting the spores of SNA1054/pSNY3.
Trp) plates containing 1 mg/ml
5-fluoroorotic acid, and colonies that grew at 23 °C but did not
grow at 37 °C were selected. The ts phenotype was
confirmed by introducing the mutagenized plasmids recovered from the
putative mutants into SNY1061-2B. Construction of the
nep98-7 strain was performed as follows. The
nep98-7 mutant gene was cloned into pBluescript II SK+, and
an 0.8-kb SmaI/StuI fragment of pJJ281 (19)
containing the TRP1 gene was introduced into the
NdeI site (blunted) of the above plasmid to yield pYT13.
pYT13 was digested with ClaI and introduced into SNY1054.
Trp+His
transformants were selected, and the
introduction of the nep98-7 mutation was confirmed by PCR.
YTE8-1B (MAT
nep98-7::TRP1
ura3 leu2 trp1 his3 suc2) and YTE8-3A (MATa
nep98-7::TRP1 ura3 leu2 trp1 his3 ade2
suc2) were constructed by dissecting the spores of the above
diploid strain, and the ts growth phenotype was confirmed.
-D-thiogalactopyranoside to the
culture. The protein was recovered as an inclusion body, solubilized
with 2 M urea, and used for immunizing rabbits.
-tubulin monoclonal antibody
(Sigma) (20). Cells were viewed on an Olympus IX-70 inverted microscope
with suitable filter sets. Images were captured by a MicroMax cooled CCD camera (Princeton Research Instruments) and analyzed by IPLab software (Scanalystics). Preparation of thin sections of yeast cells by
the freeze-substituted fixation method was carried out as described
previously (20). The sections were examined and photographed on a
JEOL2010 and H-7600 transmission electron microscope at 100 kV.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
phenotype, indicating that the NEP98 gene
is essential for yeast cell growth (data not shown). Two-hybrid
analyses with deletion mutants of Nep98p showed that residues 431-650
are essential for its interaction with Jem1p (data not shown).
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Fig. 1.
Properties of Nep98p. A,
predicted amino acid sequence of Nep98p. The hydrophobic segment is
indicated with a horizontal bar, and the putative
N-linked glycosylation sites are indicated with
asterisks. The Jem1p-interacting region is shown in
bold letters. B, localization of Nep98p. Cells of
SEY6210/pNTY5 were grown in SCD medium lacking uracil at 30 °C and
analyzed by immunofluorescence microscopy using the anti- -tubulin
monoclonal antibody. Panels a, b, and
c show the same field of the fluorescent images stained with
Nep98p-GFP, the anti-
-tubulin monoclonal antibody, and DAPI,
respectively. Bar in panel c, 5 µm.
-tubulin monoclonal antibody showed that
the bright dots represent the ends of the spindle microtubule (Fig.
1B, panel b). These results suggest that Nep98p
is a nuclear envelope protein and is enriched in the SPB. Subcellular
fractionation followed by immunoblotting with the anti-Nep98p
antibodies showed that Nep98p was recovered in the microsome fraction
(data not shown). The nuclear envelope localization of Nep98p was not
affected by the nup133 mutation, which causes clustering of
the nuclear pore complex (22), suggesting that Nep98p is not a
component of the nuclear pore complex (data not shown).
TX-100, Fig.
2B), the C-terminal part of Nep98p, including the Jem1p-interacting domain, is exposed to the ER lumen. Endoglycosidase H
treatment of the microsome fraction converted Nep98p, which contains
three C-terminal putative N-glycosylation sites (residues 278, 303, and 304; Fig. 1A), to a 93-kDa fragment (data not
shown), supporting the fact that Nep98p is a type II integral membrane protein.
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Fig. 2.
Nep98p is an integral membrane protein that
interacts with Jem1p. A, microsomal membranes prepared
from the strain SEY6210 were incubated with the following solutions on
ice for 30 min and subsequently centrifuged at 315,000 × g for 30 min: buffer, buffer (10 mM
Tris-HCl, pH 7.4) alone; NaCl, buffer containing 1 M NaCl; urea, buffer containing 2 M urea; TX, buffer containing 1% Triton X-100;
TX + NaCl, buffer containing 1 M NaCl
and 1% Triton X-100;
Na2CO3, 0.1 M
Na2CO3, pH 11.5. Pellets (P) and
supernatants (S) derived from the same amount of microsomes
(T) were subjected to SDS-PAGE followed by
immunoblotting with the anti-Nep98p antibodies. B,
microsomal membranes prepared from the strain SEY6210 were incubated
with 0.5 mg/ml trypsin in the absence ( TX-100) or presence
(+TX-100) of 0.1% Triton X-100 on ice for the indicated
times. Digestion was stopped by the addition of soybean trypsin
inhibitor to 1 mg/ml, and the proteins were analyzed by SDS-PAGE and
immunoblotting with the anti-Nep98p antibodies. C,
spheroplasts prepared from the strain SEY6210/pSNY6, pSNJ21 were
labeled with 35S-amino acids. The spheroplasts were
disrupted with 0.1% Triton X-100 and incubated with DSP at the
concentrations shown above the lanes for 20 min on ice. Proteins were
extracted and subjected to immunoprecipitation with the anti-Nep98p
antibodies. Immunoprecipitates were incubated with 10 mM
dithiothreitol and subjected to the second round of immunoprecipitation
with the anti-Nep98p antibodies (98) or the anti-HA
monoclonal antibody (HA).
Lys in the Jem1p-interacting domain of Nep98p.
We constructed a nep98-7 mutant strain by introducing the
Asn597
Lys mutation into the chromosomal
NEP98 locus for further analyses. When nep98-7
cells were shifted to the restricted temperature of 37 °C, the
fraction of large budded cells increased to 67%, whereas the fraction
of small budded cells decreased to 9% (Table I). This phenotype is characteristic
of yeast mutants that have defects in the G2/M progression.
We also observed an increase in the cell size of nep98-7
cells at both 23 and 37 °C (data not shown).
The nep98-7 mutant is defective in G2/M progression
70% of large budded
wild-type cells contained two nuclei, and
30% contained a dividing
nucleus at both 23 and 37 °C (Table II).
In contrast, only 36% of large budded nep98-7 cells
contained two nuclei at 23 °C, and this fraction decreased to 8% at
37 °C. The fraction of large budded nep98-7 cells
containing one nucleus was 43 and 59% at 23 and 37 °C,
respectively. Nuclei frequently were observed near the bud neck in
these cells, but 17-19% of the mononucleate cells showed asymmetric
nuclear staining (arrow, Fig.
3d) at both 23 and 37 °C.
Similar defects in the nuclear division also were observed when Nep98p
was overexpressed from the GAL1 promoter (data not
shown).
The nep 98-7 mutant is defective in nuclear division
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Fig. 3.
Aberrant microtubule morphology observed in
the nep98-7 mutant. Wild-type (SEY6211,
a and b) and nep98-7 mutant (YTE8-3A,
c and d) cells were shifted from 23 to 37 °C,
incubated for 4 h, and subjected to preparation for
immunofluorescence microscopy using the anti- -tubulin antibody. The
panels show fluorescent images stained with the
anti-
-tubulin antibody (a and c) and with DAPI
(b and d). Arrowhead in panel
c, cytoplasmic focus of microtubules; arrow in
panel d, nucleus with asymmetric nuclear staining;
bar in panel d, 5 µm.
-tubulin antibody. In
wild-type cells, two nuclei were located on the distal sides of both of
the dividing cells, and the spindle microtubules were elongated with
their axis parallel to the long axis of the cell (Fig. 3a).
On the other hand, in nep98-7 cells, short spindles were
observed in mononucleate large budded cells, and their axis was not
parallel to the long axis of the cell (Fig. 3c). We also
observed aberrant microtubule organization in nep98-7 cells
at 37 °C. A significant portion of the nep98-7 cells
contained a focus of microtubule staining that was not in close contact
with the DNA depicted by DAPI staining (arrowhead, Fig.
3c). These results indicate that nep98-7 cells have defects in the spindle organization during nuclear division.
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Fig. 4.
Electron micrographs of the
nep98-7 mutant. Wild-type (SEY6211, A
and B) and nep98-7 mutant (YTE8-3A,
C-F) cells grown at 23 °C were prepared for electron
microscopy by the freeze-substituted fixation method. Panels
C and D show different thin sections of the same field.
G, nep98-7 mutant (YTE8-3A) cells were shifted
from 23 to 37 °C, incubated for 6 h, and subjected to
preparation for electron microscopy by the freeze-substituted fixation
method. Panels G-1-G-3 show a series of thin
sections of the region indicated with a box in panel
G. Small arrows, nuclear envelope; large
arrow in panel A, half-bridge; arrowheads,
SPBs; N, nucleus; bar in panel F, 100 nm (panels A-F); bar in panel
G, 1 µm (panel G); bar in panel
G-3, 200 nm (panels G-1-G-3).
651-682)
complemented the lethality of the nep98-null mutant at both
23 and 37 °C, the deletion of 66 C-terminal residues (
617-682) or of residues 431-460 (
431-460) within the Jem1p-interacting domain (residues 431-650) abolished the complementing ability of
Nep98p (Fig. 5A). Therefore, the Jem1p-interacting domain is essential for the Nep98p function. In contrast, two putative
coiled-coil domains in the luminal part of Nep98p (residues 241-274
and 374-401) are dispensable for the Nep98p function, as the deletion
of this segment did not affect the complementing ability of Nep98p.
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Fig. 5.
Analyses of functional domains of
Nep98p. A, schematic representation of Nep98p and its
deletion mutants investigated in this study. The dotted box,
solid box, and hatched box indicate the acidic
region, a possible membrane-spanning segment, and the Jem1p-interacting
region, respectively. The black boxes show the expressed
regions of Nep98p, and all of the deletion mutants have the 3HA epitope
tag at the N terminus (HA tag, white box).
SNY1061-1D harboring a plasmid that expresses one of the deletion
mutants of Nep98p was streaked onto SCD plates lacking tryptophan but
containing 1 mg/ml 5-fluoroorotic acid and incubated at 23 or 37 °C.
Growth phenotypes of the deletion mutants are shown on the
right. +++, cells that grew as fast as wild-type cells; ++,
cells that grew more slowly than wild-type cells; ±, cells that grew
significantly more slowly than wild-type cells; , no growth.
B, quantitative mating experiments. Wild-type or
nep98 mutant cells of the opposite mating types were mated
for 4 h at 23 °C. The mating mixtures were stained with DAPI,
and the phenotypes of the zygotes were observed by microscopy. The
numbers represent percentages of wild-type
(Kar+), nuclear fusion-defective (fusion
), or
nuclear congression-deficient (congression
) zygotes.
Mutant zygotes that contained nuclei more than 2 µm apart were scored
as the congression-deficient phenotype. At least 100 zygotes were
analyzed for each strain.
65-145) instead of expressing wild-type Nep98p could grow normally
at 23 °C but could grow only slowly at 37 °C (Fig.
5A). Deletion of 63 N-terminal amino acid residues
(
2-64) did not affect the complementing ability of Nep98p. The
effects of the deletion of the entire cytoplasmic domain of Nep98p
could not be analyzed because this deletion significantly affected the stability of Nep98p. Therefore, the N-terminal acidic domain appears to
be important for the Nep98p function at elevated temperatures.
), and 22% had nuclei
in distant positions (congression
) (Fig. 5B).
Zygotes with mutant Nep98p lacking the N-terminal acidic region
(
65-145) showed defects in nuclear fusion; 17% contained two or
more fusion
nuclei, and 28% contained
congression
nuclei. Zygotes with mutant Nep98p lacking
residues 240-430 (
240-430) were affected slightly in nuclear
fusion; 12% contained two or more fusion
nuclei.
We could not test the effects of deletion on the entire Jem1p-interacting domain because the Jem1-interacting domain is essential for normal cell growth. We thus conclude that Nep98p plays a
role in both the nuclear congression and nuclear membrane fusion steps
of karyogamy.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
jem1 zygotes are not defective in the outer nuclear
envelope fusion but are defective in the SPB fusion and the inner
nuclear envelope fusion,2
supporting this hypothesis. Because the half-bridge structure is
present on both sides of the nuclear envelope, it is possible that
Nep98p resides in both the outer and the inner nuclear envelope membranes. Further analysis of the localization of Nep98p in the nuclear envelope and the identification of Nep98p-interacting proteins
other than Jem1p will reveal the function of Nep98p in SPB functions.
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ACKNOWLEDGEMENTS |
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We thank E. A. Craig for the yeast two-hybrid system, S. Emr for the strains, the members of the Endo laboratory for discussions, and R. Swanson for critically reading the manuscript.
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Note Added in Proof |
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After acceptance of the present manuscript, we learned that Mark Winey and co-workers found the same protein (Nep98p) independently and named it Mps3p (Jaspersen, S. L., Giddings, T. H., Jr., and Winey, M. (2002) J. Cell Biol. 159, 945-956).
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a grant from the Naito Foundation.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.
¶ Research Fellow of the Japan Society for the Promotion of Science.
To whom correspondence should be addressed. Tel.:
81-52-789-2490; Fax: 81-52-789-2947; E-mail:
endo@biochem.chem.nagoya-u.ac.jp.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M210934200
2 S. Nishikawa, A. Hirata, and T. Endo, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; SPB, spindle pole body; ts, temperature-sensitive; HA, influenza virus hemagglutinin; 3HA, three tandem repeats of the HA epitope; DSP, dithiobis(succinimidyl propionate); GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole.
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