Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.
IN the yeast Saccharomyces cerevisiae, the nuclear envelope remains intact during all stages of mating. The
diploid nucleus is generated from the direct fusion of
two haploid nuclei in a process called karyogamy (for review see Rose, 1996 Nuclear envelope fusion during karyogamy is a prime
example of homotypic membrane fusion. Homotypic (or
self) fusion involves the union of two separate membranes
of the same composition and origin (for review see Rothman and Warren, 1994 Genetic studies of yeast have identified many genes that
mediate the three major pathways of membrane fusion:
(a) secretory vesicles with target organelle membranes
(Novick et al., 1980 During both nuclear congression and nuclear fusion, the
yeast microtubule organizing center, the spindle pole body,
plays a central role (Byers and Goetsch, 1975 Genes necessary for karyogamy have been identified in
genetic screens for kar mutants (Conde and Fink, 1976 Recently, a novel screen has been used to isolate bilateral karyogamy mutants (Kurihara et al., 1994 Since nuclear congression is a microtubule-dependent
process (Delgado and Conde, 1984 The Class II nuclear fusion mutants are defined by the
genes KAR2, KAR5, KAR7, and KAR8. Class II mutants
exhibit defects in membrane fusion that are evident in
vivo, as observed by electron microscopy (Kurihara et al.,
1994 The best characterized of the Class II genes is KAR2,
which encodes an ER-localized HSP70. Kar2p is the yeast
homologue of the mammalian protein BiP/GRP78 (Normington et al., 1989 In this paper, we describe the cloning and characterization of the Class II karyogamy gene KAR5. KAR5 encodes
a novel protein that plays a central role in nuclear membrane fusion. KAR5 is induced during mating, and Kar5p
localizes to the initial site of nuclear fusion, next to the
spindle pole body. Analyses of Kar5p indicated that it is
membrane bound, with most of the protein positioned
within the lumen of the ER/nuclear envelope. Since it is
sequestered completely within the ER, Kar5p is probably not a membrane fusogen or a receptor linking the two nuclei for docking. However, Kar5p may help determine
when and where nuclear membrane fusion occurs or may
be required to complete membrane fusion once nuclear
contact has been made. As such, Kar5p represents a novel
protein required for the regulation and activation of homotypic membrane fusion.
Microbial Techniques and Strain Constructions
Culture media and genetic manipulations were as described elsewhere
(Rose et al., 1990 Quantitative matings were performed as previously described (Rose et al.,
1990 Table I.
Yeast Strains Used
). Karyogamy proceeds in two steps:
nuclear congression and nuclear fusion. Nuclear congression follows immediately after cell fusion when the haploid nuclei are drawn together in the newly formed zygote. Once the nuclei are in close proximity, the two nuclear envelopes fuse, generating a diploid nucleus.
). Examples of self fusion in animal
cells include the reassembly of the nuclear envelope (Wiese
and Wilson, 1993
) and the Golgi apparatus (Acharya et al.,
1995
; Rabouille et al., 1995
) from membrane fragments during mitotic telophase. Homotypic fusion is also an inherent and essential step in secretion. Vesicle budding, like
other processes of fission, requires homotypic membrane
fusion. To be released from an organelle, homotypic membrane fusion is required to "pinch off" the constriction
around the base of each secretory vesicle. It is likely that
the study of yeast karyogamy will help identify conserved
factors involved in all processes of homotypic fusion (Rothman, 1994
; Rothman and Warren, 1994
).
), (b) plasma membrane fusion during
mating (McCaffrey et al., 1987
; Trueheart et al., 1987
; Berlin et al., 1991
; Kurihara et al., 1994
), and (c) nuclear envelope fusion (Conde and Fink, 1976
; Polaina and Conde,
1982
; Kurihara et al., 1994
). The mating-specific processes
of cell fusion and karyogamy are solely events of homotypic membrane fusion. For recent reviews see Rose
(1996)
and Marsh and Rose (1997)
.
; Rose, 1991
;
Rose 1996
). The disk-shaped spindle pole body of S. cerevisiae is embedded in the nuclear envelope and serves an
analogous function to that of the centrosome in larger eukaryotes (Winey and Byers, 1993
). The spindle pole body
assembles both the mitotic and meiotic spindles as well as
cytoplasmic microtubules. The spindle pole body plays two roles in karyogamy. During nuclear congression, the
two spindle pole bodies anchor the cytoplasmic microtubules by which the two haploid nuclei are drawn together.
During nuclear fusion, the spindle pole body marks the
initial site of both membrane fusion and fusion of the two
spindle pole bodies (Byers and Goetsch, 1975
). As the two
spindle pole bodies join, the two respective outer and inner nuclear envelopes fuse. Thus, nuclear fusion is a complex and coordinated series of events. It is not known
whether nuclear membrane fusion proceeds by a single
concerted fusion event or by a series of discrete events involving fusion of first the outer and then the inner membranes.
;
Polaina and Conde, 1982
; Kurihara et al., 1994
). The first kar
mutant screens identified mutations that caused karyogamy defect even when the mutants were mated with wild-type cells (Conde and Fink, 1976
; Polaina and Conde, 1982
).
Such mutants were referred to as "unilateral" in that the
defect was evident when a single mating partner contained the mutation. Both dominant and recessive unilateral mutants have been isolated. One explanation for the recessive
unilateral mutants is that, in the zygote, the mutant nucleus cannot receive the corresponding wild-type protein
from the wild-type nucleus. Presumably this results when
the wild-type Kar protein is restricted to the wild-type nucleus and is not free to diffuse and rescue the mutant defect of the other nucleus before they both enter mitosis
(Rose et al., 1989
). In contrast, in bilateral karyogamy defects both partners in a mating must be mutant to observe
the mutant phenotype. In the bilateral mutants, the gene
products must fulfill one of the following criteria: (a) be
diffusible, (b) be required in only one nucleus, or (c) be
synthesized in the zygote such that both nuclei acquire sufficient nascent wild-type protein.
). These new
mutants identified the karyogamy genes KAR4, KAR5,
KAR7, KAR8, and KAR9. Based on cytological and genetic criteria, all kar mutants were grouped into two functional classes (Kurihara et al., 1994
). Class I mutants exhibit a block in nuclear congression: the nuclei do not fuse and remain distant from one another in the zygote. Class
II mutant zygotes exhibit closely juxtaposed but unfused
nuclei, consistent with a block in nuclear membrane fusion.
; Rose and Fink, 1987
;
Huffaker et al., 1988
; Berlin et al., 1990
; Rose, 1991
), it is
not surprising that most Class I mutants represent genes
that affect microtubule function: KAR1 encodes a component of the spindle pole body (Vallen et al., 1992
; Spang
et al., 1995
); TUB2 encodes
-tubulin (Huffaker et al.,
1987
, 1988
); BIK1 encodes a microtubule-associated protein (Berlin et al., 1990
); KAR3 encodes a kinesin homologue (Meluh and Rose, 1990
); CIK1 encodes a Kar3p-
associated protein (Page and Snyder, 1992
; Page et al.,
1994
); and KAR4 regulates the mating-induced transcription of KAR3 and CIK1 (Kurihara et al., 1996
). Microtubules and associated proteins play similar roles in both
karyogamy and in the condensation of vesicular ER/nuclear fragments in animals (Dabora and Sheetz, 1988
). Microtubules allow membranes attached to them to be concentrated, thereby promoting their fusion.
; Beh, 1996
), and in vitro, as measured in a homotypic
ER/nuclear membrane fusion assay (Kurihara et al., 1994
;
Latterich and Schekman, 1994
). The in vitro ER/nuclear membrane fusion assay indirectly measures membrane fusion by detecting the consequences of lumenal mixing. When
fusion occurs, a secretory precursor protein preloaded into
the lumen of one ER membrane can be processed by a
glucosidase resident only in the lumen of a second ER
membrane. Thus, the assay is sensitive to mutations that
block both the initiation as well as the completion of membrane fusion. In contrast to the Class II mutants, ER membranes derived from the Class I mutant strains fused as efficiently as wild-type membranes in vitro.
; Rose et al., 1989
). In addition to its role
in karyogamy, Kar2p is essential for the translocation of
secretory precursors into the ER (Vogel et al., 1990
).
Given the known activities of Kar2p and its homologues,
Kar2p could have several different roles in karyogamy. Kar2p may be indirectly required for nuclear fusion though
its role in the translocation and/or folding of a key component of the nuclear fusion apparatus. Alternatively, Kar2p
may be necessary to maintain the structure of the ER/nuclear envelope during nuclear fusion. However, a variety
of genetic and biochemical data (Vogel et al., 1990
; Latterich and Schekman, 1994
) argue that it is more likely that
KAR2 is directly involved in karyogamy. In this regard, Kar2p may be functioning as a molecular chaperone, participating in the assembly or interactions of a nuclear membrane fusogenic complex. Such a complex may be comprised of the gene products of some of the other kar genes.
Materials and Methods
). Cells treated with
-factor were incubated in the presence of 10 µg/ml
-factor for 2.5 h in synthetic complete (SC)1 media
brought to pH 3.5 with HCl. Pheromone induction was verified microscopically when >80% of the cells formed mating projections.
). For cytoductant analysis, cyhR/rho0 cells were obtained as previously described (Rose et al., 1990
). Yeast strains used are listed in Table I.
KAR5 Cloning, Mapping, and Sequencing
Restriction enzymes used for cloning were obtained from New England
Biolabs (Beverly, MA). PCR and sequencing primers were synthesized at
the Princeton University oligonucleotide synthesis facility. Cloning techniques were performed as described in Sambrook et al. (1989). Plasmids
were recovered from yeast by the method of Hoffman and Winston (1987)
and transformed into Escherichia coli by electroporation.
To map KAR5 on the yeast physical map, the 2.8-kb HindIII subclone
from pMR2710 was labeled by the random primer method (Sambrook et al.,
1989) and hybridized to lambda prime clone filters provided by Linda
Riles (Washington University School of Medicine, St. Louis, MO).
To sequence the 2.8-kb KAR5 minimal complementing region, the genomic subclone was inserted into the unique HindIII polylinker site of
Bluescript KS+ and KS (Stratagene, La Jolla, CA). Exonuclease III/mungbean nuclease digestions were performed according to Henikoff (1987)
.
The resulting deletion clones were then sequenced using either T3 or T7
sequencing primers. Both strands of the 2.8-kb subclone were sequenced
by single-strand sequencing (Sambrook et al., 1989
).
Plasmid Constructions
To disrupt the KAR5 gene, two disruption alleles were generated, each
with a different selectable marker for integration. The first disruption allele (pMR2741) was made through the ligation of the HindIII/KpnI and
SacI/HindIII insert fragments of pMR2710 into the KpnI/SacI polylinker
sites of pRS406 (Sikorski and Hieter, 1989). To integrate and disrupt the
KAR5 open reading frame, pMR2741 was digested with HindIII, and the
entire linearized plasmid was transformed into yeast. This disruption allele was marked with the URA3 gene and transformants were selected on
SC-Ura. The second disruption plasmid (pMR2869) was constructed by ligation of the 590-bp HindIII/AvrII and 1,100-bp XhoI/HindIII fragments
of pMR2710 into the XbaI/XhoI polylinker sites of pRS405, a LEU2-marked integration plasmid. Plasmid pRS405 is the equivalent of pRS305
(Sikorski and Hieter, 1989
), with the polylinker in the opposite orientation with respect to the plasmid. To disrupt the KAR5 locus, pMR2869 was digested with HindIII, and yeast transformants were selected on SC-Leu. Cells bearing the kar5 disruption alleles were tested to show that
CEN-based vectors bearing KAR5 [pMR2710] suppressed the kar5-
mating defects.
To construct a vector for the overexpression of KAR5 gene, the 2.8-kb
HindIII fragment from pMR2710, bearing the KAR5 minimal complementing region, was subcloned into the HindIII sites of the 2-µm vector
pRS424. This vector is equivalent to pRS306 (Sikorski and Hieter, 1989),
except that the polylinker is in the opposite orientation and a segment of
the yeast 2-µm plasmid has been inserted at the AatII site. At the same
time, an equivalent centromeric plasmid was constructed using pRS426.
Plasmid pRS426 is equivalent to pRS316 (Sikorski and Hieter, 1989
), with
the exception that the polylinker is in the opposite orientation. As assayed
by interrupted plate matings (Rose et al., 1990
), both the 2-µm plasmid,
pMR3142, and the centromeric plasmid, pMR3179, suppressed the karyogamy defect of the kar5-
2 strain MS3577. Wild-type cells (MS52) transformed with pMR3179 or pMR3142 had no observable defects with respect to mating efficiency or viability (at 23, 30, or 37°C) and pMR3179 did not suppress the karyogamy defect of the other Class II karyogamy mutants (as tested by interrupted mating assays).
To construct a glutathione-S-transferase (GST)-Kar5p fusion protein
vector for expression of Kar5p in bacteria, a fragment of KAR5 corresponding to the NH2-terminal 345 residues was amplified by PCR using
primers 5GTC-GGG-ATC-CCA-TGT-TTG-AAA-TGC-TCT-ACG3
and 5
CCG-AGG-ATC-CTT-CCA-TTT-GTG-ATT-CAG3
. These primers had BamHI sites incorporated into the ends to allow the in-frame ligation of the amplified product into the BamHI site of the vector pGEX-1
(Smith and Johnson, 1988
), thus producing the plasmid pMR2739. The
corresponding transformed bacterial strain was used to make recombinant
protein to generate polyclonal antisera.
RNA Analysis
For RNA-blots, total RNA was isolated, electrophoresed through 0.8%
agarose formaldehyde gels, and transferred to GeneScreen hybridization
transfer membrane (NEN Research Products, Boston, MA) as described
elsewhere (Rose et al., 1990). Hybridization and washes were performed
as follows. Blots were prehybridized for 4 h at 68°C in 5× PPiESS buffer
(0.75 M NaCl, 5 mM Na4P2O7, 0.5 M Na2EDTA, 0.125 M sodium phosphate, pH 8.0) containing blocking solution (0.2% ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.1 µg/ml denatured salmon sperm DNA,
and 1.0% SDS. Boiled probe was added directly to the blots in prehybridization mix and incubated overnight at 68°C. Blots were rinsed first in 0.2×
PPiESS, 0.5% SDS for 15 min and then washed in the same buffer twice,
each time for 20 min at 68°C.
Probes were labeled by the random primer method (Sambrook et al.,
1989). The KAR5 probe used was derived from the KAR5 2.8-kb HindIII
fragment isolated from pMR2710. As an internal control, levels of KAR5
mRNA were compared with levels of ACT1 transcript. The probe used for
hybridization to ACT1 mRNA was derived from a 282-bp ACT1 EcoRI/
HindIII fragment isolated from pYST122 (a gift from J. Broach, Princeton
University, Princeton, NJ).
Antibody Production and Purification
Antisera against bacterially expressed GST-Kar5p was generated in a
New Zealand white rabbit at the Princeton University Animal Facility.
GST-Kar5p was purified from inclusion bodies from four 500-ml cultures
of MR2739 incubated with 0.5 mM IPTG for 4 h at 37°C. Bacterial pellets
were resuspended in 1/50 culture volume of 16 mM Na2HPO4, 4 mM
NaH2PO4, 150 mM NaCl, 1% Triton X-100 containing 2 µg/ml final concentration each of chymostatin, leupeptin, aprotinin, pepstatin A (Sigma
Chemical Co., St. Louis, MO), and AEBSF (Calbiochem Corp., La Jolla,
CA). Cells were disrupted by three 1-min pulses of a sonicator. After centrifugation at 12,000 g at 4°C, the pellet was resuspended in Laemmli sample buffer (Laemmli, 1970) and boiled for 15 min. GST-Kar5p was resolved on a 10% SDS-PAGE and transferred to nitrocellulose (Schleicher
& Schuell, Inc., Keene, NH), and the band corresponding to GST-Kar5p
was excised. The nitrocellulose strip, bearing ~0.2 mg of Kar5p, was dissolved in 0.2 ml DMSO, mixed with an equal volume of sterile water,
emulsified with 0.5 ml Freund's Complete Adjuvant (Sigma Chemical
Co.), and injected into rabbits. Boosts were performed every month with
100 µg GST-Kar5p (either on nitrocellulose dissolved in DMSO or isolated by preparative electrophoresis [see below]) emulsified in Freund's
Incomplete Adjuvant (Sigma Chemical Co.).
To affinity purify anti-Kar5p antibodies, GST-Kar5p was isolated by preparative electrophoresis and then covalently coupled to a chromatographic resin. After preparative 10% SDS-PAGE, the GST-Kar5p band was identified by negative staining with 0.3 M CuCl2. The protein was electroeluted from the excised gel slice at 100 V for 16 h in Tris-glycine buffer (25 mM Tris, 200 mM glycine) between BT1 and BT2 Elutrap membranes (Schleicher & Schuell, Inc.). 0.5-1.0 mg of the protein (as determined by Bradford assay [1976]) was conjugated to Sulfo-link gel (Pierce Chemical Co., Rockford, IL), as suggested by the manufacturer (except 1 mM DTT was used instead of 2-mercaptoethylamine). 1 ml of antisera was applied to the column, the column was washed, and anti-Kar5p-specific antibodies were eluted with 0.1 M glycine, pH 2.5.
Immunological Techniques
Yeast extracts for Western blots were prepared as described by Ohashi et al.
(1982). For Western blotting, proteins were separated by SDS-PAGE and
transferred in 20 mM Tris, 150 mM glycine, 20% methanol to nitrocellulose (Schleicher & Schuell, Inc.). Immunoblots were blocked in 5% nonfat
milk in buffer A (10 mM Tris-HCl pH 7.4, 150 mM NaCl) for 1 h at room
temperature. Blots were then incubated for at least 2 h at room temperature, with primary antibody diluted in 5% milk/buffer A. Before incubations with the secondary antibody, blots were washed four times for 15 min each in buffer A, 0.05% Tween-20 (Sigma Chemical Co.) at room
temperature. HRP-conjugated anti-rabbit secondary antibody (Amersham Corp., Arlington Heights, IL), diluted in 5% milk/buffer A, was
added to blots for 1 h, after which washes were performed as before.
Before use, affinity-purified anti-Kar5p antibodies were preadsorbed
to kar5- cells as follows: 150 µl of affinity-purified antibodies were incubated with 200 µl of packed, formaldehyde-fixed kar5-
2 cells (MS3980)
for 2 h at 23°C. Cell walls were removed from the fixed cells by prior incubation at 30°C with 50 µl of
-mercaptoethanol and 0.5 ml 10 mg/ml Zymolyase 100,000 T in 1.2 M sorbitol. The antibody/cell mixture was then
centrifuged in a microcentrifuge, and the supernatant was incubated with
a second volume of fixed kar5-
2 cells. After centrifugation, supernatant was collected, and the preadsorbed antibody was stored at 4°C with 0.2%
NaN3 as a preservative.
For Western blots, affinity-purified preadsorbed anti-Kar5p antibody was used at a 1:100 dilution, rabbit anti-Kar2p antiserum was used at a titer of 1:5,000, rabbit anti-Sec72p serum was used at a dilution of 1:1,000 (a gift of Martin Latterich, University of California, Berkeley, CA), and rabbit anti-Sec61p antibodies were used at a dilution of 1:1,000 (a gift of Vladimir Lupashin, Princeton University, NJ). Bands were visualized with a 1:3,000 dilution of the HRP-conjugated anti-rabbit secondary antibody, followed by the ECL chemiluminescent system (Amersham Corp.).
Isolation of Nuclei and Protease Protection
Nuclei were isolated as described by Aris and Blobel (1991) with minor
modifications. 30,000 Klett units of cells, preincubated with 0.1 mg/ml
-factor for 2.5 h, were harvested for nuclear isolation. Cells were rinsed
and pelleted twice and resuspended in 10 ml spheroplast buffer (0.1 M potassium phosphate, pH 6.5, 1.2 M sorbitol, 1 mM PMSF). To remove cell
walls, cells were treated with 50 µl of
-mercaptoethanol and 0.5 ml 10 mg/ml Zymolyase 100,000 T. After 45 min of digestion, spheroplasts were
recovered by centrifugation at 2.5 K rpm in a clinical centrifuge (model GLC2B; Sorvall, Newtown, CT). After two rinses with spheroplast buffer,
spheroplasts were resuspended in 25 ml lysis buffer (20% Ficoll 400, 20 mM
potassium phosphate, pH 6.5, 1 mM MgCl2, 1 mM PMSF). Spheroplasts
were gently lysed with 20 strokes of a stainless steel Dounce homogenizer.
Cellular disruption was confirmed by microscopy. After chilling 10 min at
4°C, the lysate was centrifuged 5 min at 13,000 g (9 K rpm) in a rotor
(model SW27; Beckman Instruments, Fullerton, CA) at 4°C. The supernatant was recovered and poured into a second tube and centrifuged for 10 min at 13,000 g at 4°C.
For the protease protection assay, the supernatant from the 13,000-g centrifugation was diluted fivefold in ice-cold 20 mM potassium phosphate, 1 mM MgCl2, 1 mM PMSF, and membranes/nuclei were pelleted in 30-ml Corex tubes by centrifugation for 10 min at 11,725 g (9 K rpm) in a rotor (model SA600; Sorvall) at 4°C. Pellets were resuspended in 12 ml protease buffer (0.3 M mannitol, 0.1 M KCl, 50 mM Tris-HCl, pH 7.5, 1 mM EGTA) and divided into two portions. To one portion, Triton X-100 was added to a final concentration of 0.4%; both portions were incubated at 4°C with proteinase K, 0.3 mg/ml final concentration. 1-ml aliquots were removed at various times, and the aliquots were mixed with 0.1 ml 100% TCA at 4°C to terminate the digestion. After centrifugation in a microcentrifuge and washes with 90% acetone, protein pellets were resuspended in Laemmli sample buffer and boiled, and proteins were resolved by 10% SDS-PAGE.
To obtain purified nuclei, the cleared lysate was fractionated further
(Aris and Blobel, 1991). The supernatant obtained from the 13,000-g spin
was split and layered on top of two SW27 tubes containing step gradients
of 5 ml 50, 40, and 30% Ficoll 400 (in 20 mM potassium phosphate, pH 6.5, 1 mM MgCl2, 1 mM PMSF) layered from bottom to top, respectively. The
tubes were centrifuged in a rotor (model SW27; Beckman Instruments) at
18 K rpm for 1 h at 2°C. Nuclei were isolated from the 40% Ficoll layer
(including the 40-50%, and 30-40% interfaces), diluted 10-fold with 20 mM potassium phosphate, pH 6.5, 1 mM MgCl2, 1 mM PMSF, and pelleted by
centrifugation in 30-ml Corex tubes for 10 min at 9 K rpm in a rotor
(model SA600; Sorvall) at 4°C. Nuclei were resuspended in 50 mM Tris-HCl, pH 7.0, 1 mM PMSF. Total cellular protein was precipitated by the
method of Ohashi et al. (1982)
, and from the fractionated nuclei, proteins
were precipitated with the addition of TCA to a 10% final concentration.
Pellets were rinsed with 90% acetone and resuspended by boiling in
Laemmli loading buffer without bromophenol blue or
-mercaptoethanol;
protein concentrations were then determined by a Lowry-based protein
assay (Bio-Rad DC protein assay; Bio-Rad Laboratories, Inc., Hercules, CA).
Finally,
-mercaptoethanol was added (2% final concentration) with bromophenol blue and equal amounts of samples were run on a 10% SDS gel.
To determine whether Kar5p was a membrane protein, extractions were performed with 1 M NaCl, 0.1 M Na2CO3, pH 11.0, 2 M urea, 1% Triton X-100, and 1% Triton X-100 plus 0.5 M NaCl. A crude membrane fraction was isolated as described above and resuspended in 20 mM Tris-HCl, pH 7.4 with protease inhibitors (chymostatin, leupeptin, aprotinin, pepstatin A, AEBSF, and PMSF). To this fraction, NaCl, detergent, or Na2CO3 was added, and after a 1-h incubation on ice, the samples were centrifuged at 128,000 g (model TL-100 ultracentrifuge, model TLA 100.2 rotor; Beckman Instruments) for 1 h. Pellets were resuspended, and equal volumes of sample were electrophoretically separated by SDS-PAGE.
Immunofluorescence Microscopy
Indirect immunofluorescence, using paraformaldehyde fixation of intact
cells, was performed with modifications of the procedure described by
Roberts et al. (1991). Harvested cells were washed with 0.1 M potassium
phosphate, pH 6.5, pelleted in a clinical centrifuge, and resuspended in 2 ml
fresh paraformaldehyde solution (2 g paraformaldehyde, 350 µl 6 N
NaOH, 50 µl 1 M MgCl2, and 0.68 g KH2PO4 in 50 ml distilled H2O) without pretreatment with formaldehyde. Cells were allowed to fix overnight
at 23°C. The fixed cells were washed twice in 0.1 M potassium phosphate,
pH 6.5, 1.2 M sorbitol and then resuspended in 1 ml of the same solution.
To remove cell walls, fixed cells were incubated with 5 µl
-mercaptoethanol, 25 µl glusulase (DuPont Biotechnology Systems, Boston, MA), and 15 µl 10 mg/ml Zymolyase 100,000 T at 30°C for 1 h (or until they appeared dark by phase contrast microscopy). Spheroplasts were washed
once with 1.2 M sorbitol and then incubated at 23°C for 10 min with 2%
SDS, 1.2 M sorbitol. The cells were then washed twice in 1.2 M sorbitol.
Lastly, cells were affixed to glass slides coated with polylysine.
Indirect immunofluorescence using methanol/acetone fixation of spheroplasted shmoos was performed, with minor modifications, as described by
Rout and Kilmartin (1990). Spheroplasted shmoos were incubated in Wickerhams' medium for only 10 min (to avoid recovery from pheromone arrest) before being affixed onto slides. Whole cell morphology was not
preserved using this method, though it was more sensitive for Kar5p visualization.
Affinity-purified, preadsorbed rabbit anti-Kar5p antibodies were used
at a 1:10 dilution. Mouse anti-90-kD antibodies were used at a 1:500 dilution (a gift of John Kilmartin, Medical Research Council, Cambridge, England). Secondary antibodies were used at a 1:1,000 dilution (Boehringer
Mannheim Corp., Indianapolis, IN). Secondary antibodies used for the
detection of Kar5p were rhodamine-conjugated goat anti-rabbit or Cy3-conjugated goat anti-rabbit; anti-90-kD secondary antibodies were FITC-conjugated goat anti-mouse. 4,6-diamidino-2-phenylindole (DAPI) was
obtained from Accurate Chemicals and Scientific Corp. (Westbury, NY).
Isolation and Mapping of KAR5
To clone the KAR5 gene, the kar5-1162 mutant strain
(MS2686) was transformed with a yeast centromere vector-
based genomic library (Rose et al., 1987). A total of 86,000 transformants (57 genomic equivalents) were screened by
replica plating onto lawns of kar5-1162 cells of the opposite mating type (MS2685) and allowing them to mate for
only 3 h before replica plating to media to select for diploids (interrupted plate matings). Several mating-proficient transformants were isolated; two of these suppressed
the kar5-1162 mating defect after isolation of the plasmids
in E. coli and retransformation back into kar5-1162 cells.
Suppression was therefore plasmid-linked, and the genomic DNA inserts on the plasmids had related restriction maps (Fig. 1).
To determine the minimal complementing region, several subclones were constructed, transformed into MS2686, and tested for mating proficiency in interrupted plate matings. The smallest complementing region, a 2.8-kb HindIII fragment, was identified (Fig. 1). The 2.8-kb HindIII subclone was used to construct KAR5 disruption alleles, which were viable and exhibited karyogamy defects similar to the original kar5 mutants. The phenotypes of the disruption alleles will be described below.
To demonstrate that the cloned DNA contained the authentic KAR5 gene, a kar5 disruption mutant was mated
to kar5-1162 and kar5-486 mutant strains (MS3258 and
MS3261, respectively). To facilitate the mating, the disruption strain contained an autonomous plasmid bearing the
putative KAR5 gene (MS3983). Selected diploids were then
grown on medium containing 5-fluoro-orotic acid (Boeke et al., 1987) to select for cells in which the KAR5 plasmid
had been lost. After sporulation and tetrad dissection, we
tested the ability of the spore clones to mate with kar5-1162 strains. From 41 tetrads, encompassing both crosses,
no Kar5+ segreagants were obtained. We concluded that
the cloned DNA was tightly linked to (
1.2 cM) and,
therefore, allelic with the authentic KAR5 locus.
KAR5 Encodes a Putative Translocated Coiled-Coil Protein
To analyze the KAR5 gene, the DNA of the smallest (2.8-kb
HindIII) complementing subclone (pMR2710) was sequenced. The complementing subclone contained only one
complete open reading frame of 1,512 bp, encoding a protein predicted to be 504 amino acids in length (Fig. 2 B)
with a molecular mass of 58.4 kD. The two disruption alleles
(pMR2741 and pMR2869) delete large segments of this
open reading frame (see Fig. 1), and neither complemented the kar5-1162 karyogamy defect. Using the 2.8-kb
subclone as the probe, the KAR5 gene was physically
mapped by hybridization with whole chromosome blots
and lambda prime clone filters (Riles et al., 1993) to chromosome XIII near the ADH3 gene. The gene has also been
sequenced by the Saccharomyces genome sequencing project and corresponds to hypothetical protein YMR065W.
The two sequences of KAR5 concur, with the exception of
1 bp difference at residue 5. Our sequence predicts a leucine at this site, whereas the sequencing project predicts an
arginine.
In the smallest complementing subclone, pMR2710, 259 bp of sequence upstream of the KAR5 coding region was present together with the entire KAR5 open reading frame (Fig. 2 A). Since this subclone fully complemented the karyogamy defect of kar5 mutants, we conclude that all the sequence elements required for KAR5 function are contained within the 259-bp upstream sequence. Contained within this region are two putative pheromone response elements (PREs). PREs are the binding site for Ste12p, the transcription factor that controls genes induced in mating. Both putative PREs match the consensus sequence in seven out of eight residues.
Three regions of the Kar5p protein were predicted to be
significantly hydrophobic (Figs. 2 B and 3). The first corresponds to the amino-terminal 20 residues and may therefore correspond to a secretory signal sequence. However,
since no sites were found that conform to the consensus
-3,-1 rule for a signal peptide cleavage site (von Heijne,
1986), if the NH2-terminal sequence does function as is a
signal peptide, it may not be cleaved. Two other regions
predicted to form significantly hydrophobic regions were
found towards the carboxyl terminus (residues 455-470
and 485-502), and these may form transmembrane spanning domains, although no strong predictions about the
orientation can be made (Hofmann and Stoffel, 1993
; Rost
et al., 1995
). Taken together, the primary structure suggests that Kar5p enters the secretory pathway and is an integral membrane protein with one or more carboxy-terminal regions spanning the membrane.
Two other predicted regions of the protein are noteworthy. First, Kar5p is predicted to contain a region with significant probability of forming two coiled-coil domains
(Lupas, 1996; Fig. 3 C). Through these two domains, Kar5p
may interact with itself or other proteins. Second, the carboxy-terminal domain of Kar5p is extremely basic. The
overall isoelectric point (pI) of the entire protein was predicted to be 4.59, and the pI of the first 350 residues was
4.24. In contrast, the pI of carboxy-terminal 150 residues
was predicted to be 10.07. Indeed, between residues 415 and 500, there are 14 basic residues and only 1 acidic residue. The functional significance of the basic COOH-terminal domain of Kar5p is uncertain, but its potential interaction with the acidic head groups of phospholipids merits
consideration.
Although KAR5 does not have close relatives in the
public databases, it does share significant homology with a
newly identified gene from Schizosaccharomyces pombe,
tht1. (These sequence data are available from Genbank/
EMBL/DDBJ under accession number D87337.) The tht1
gene is predicted to encode a 62.9-kD protein with 578 residues. Kar5p and tht1p share three regions of significant
homology (Fig. 3 D) containing 17 amino acids (33% identity, 83% similarity), 56 amino acids (28% identity, 61%
similarity), and 23 amino acids (41% identity, 54% similarity). Like KAR5, the S. pombe gene encodes a potential
membrane protein with as many as four hydrophobic regions that are predicted to form transmembrane spanning
domains. Like Kar5p, tht1p is predicted to contain a region of -helical coiled-coil. Finally, like Kar5p, the carboxy-terminal region of tht1p is predicted to be extremely
basic; between residues 392 and 504 there are 14 basic and
no acidic residues. As in Kar5p, the basic region corresponds to the putative transmembrane spanning domains.
Finally, the Genbank notation for tht1 includes the statement that the gene is required for nuclear fusion in S. pombe, although no evidence for this has been published.
Given their similarity of proposed structure and function,
it seems likely that these two proteins are homologues.
kar5- Has a Bilateral Karyogamy Defect
Karyogamy mutants can be classified as unilateral or bilateral. Unilateral mutants are defective when mated with
wild-type cells; in bilateral mutants, both partners in a
mating must be mutant to manifest a defect. The two original mutant alleles of KAR5, kar5-486 and kar5-1162, were
identified in the screen for bilateral karyogamy mutants
(Kurihara et al., 1994). To determine if the bilateral karyogamy defect reflected the true null phenotype, kar5-
mutants were constructed, and the zygotes were examined by
microscopy and quantitative mating assays (Table II). The
cloned DNA encoding the putative KAR5 gene was used
to disrupt the corresponding open reading frame (Fig. 1).
The disruption, marked with the LEU2 gene (kar5-
2) and
integrated into a wild-type haploid strain, had no effect on
cell viability at any temperature. The percentage of mutant zygotes was determined by microscopy (Table II) for matings involving kar5-
1 and kar5-486 strains. In bilateral or self-matings, the kar5-
1 mutants exhibited a pronounced karyogamy defect, similar to if not greater than
that of kar5-486. In crosses with a wild-type strain (unilateral matings), both kar5-
1 and kar5-486 manifested slight
karyogamy defects, indicating that wild-type Kar5p from
one parent is usually sufficient to effect nuclear fusion. As
was found for kar5-486 and kar5-1162, the nuclei were
closely apposed in all of the kar5-
1 zygotes exhibiting the
Kar
defect. These results confirm that Kar5p has no function in nuclear congression.
Table II. Quantitative Mating Experiments |
Measurement of the formation of cytoductants provides
a genetic assay for the failure in nuclear fusion. Cytoductants are the aberrant progeny that arise from Kar zygotes. They contain the haploid nucleus of one mating parent but cytoplasmic elements (e.g., mitochondria) from the
other parent. The cytoductants arising from the cross can
be measured using a combined selection for the functional
mitochondria coming from one parent and for the recessive drug resistance marker (cycloheximide resistance) from
the nucleus of the other parent. The calculated fraction of
cytoductants to the total number of diploids provides a
sensitive measure of a karyogamy defect (Conde and Fink,
1976
). Compared to the wild type, kar5-
2 strains displayed about an 11-fold decrease in diploid formation
from bilateral matings (Table II). This corresponded to a
roughly 3,600-fold increase in the frequency of cytoductants relative to the isogenic wild type (Table II). In unilateral matings, kar5-
2 strains formed cytoductants ~70-fold more frequently than the wild-type strains. However, this amounts to a failure in nuclear fusion occurring in
<5% of the matings. In contrast, viable cytoductants are
formed almost twice as frequently as diploids in the bilateral matings. Because viable cytoductants are not formed
in every case of a failure in nuclear fusion (Rose, 1991
), it
is likely that the failure rate is actually higher, similar to
that seen by direct microscopy of the zygotes. The kar5-
2
bilateral defect was comparable to the defects observed
for the original unilateral KAR mutants (Conde and Fink,
1976
; Polaina and Conde, 1982
). However, this defect is
not as severe as that observed in kar3-
bilateral matings (Meluh and Rose, 1990
), suggesting that a secondary pathway for nuclear envelope fusion may exist.
KAR5 Expression Is Induced by -Factor
The PRE sequence motif found upstream of most pheromone-inducible genes mediates transcriptional induction
in response to activation of the pheromone-activated protein kinase pathway by mating factor. Because of the
PREs observed upstream of KAR5, we tested directly
whether the expression of KAR5 was induced upon pheromone treatment. Total RNA was isolated from strains before and after -factor induction, and equal amounts were
analyzed by RNA-blot hybridization (Fig. 4). The observed size of the KAR5 mRNA, 2.1 kb, was consistent
with the predicted length of the KAR5 open reading
frame. KAR5 mRNA was present at low levels in vegetatively growing cells (more readily apparent after longer exposures of the autoradiograph) and was markedly induced after
-factor treatment (Fig. 4, lane B). In a ste12-
strain, the level of KAR5 mRNA did not increase, indicating that induction required the STE12 transcription factor
and the mating-response signal transduction pathway. The
vegetative level of KAR5 transcription was not higher in
diploids and was not present in kar5-
strains (data not
shown). Induction by mating pheromone is consistent with
a specific role for Kar5p in nuclear fusion during mating.
Kar5p Is a Nuclear Envelope Protein
To characterize the Kar5 protein, we used GST-Kar5p expressed in E. coli to produce polyclonal antibodies that
were then affinity-purified (see Materials and Methods).
To detect Kar5p, total protein was extracted from cells
overexpressing KAR5 from a multicopy plasmid, with and
without pheromone induction. On protein blots, the Kar5p antibodies recognized a 58-kD protein after pheromone
induction, corresponding to the predicted size of unmodified Kar5p (58,396 D). In contrast, the 58-kD protein was
not detectable after pheromone induction in a strain bearing a deletion of the KAR5 gene (MS3986) (Fig. 5). Taken
together, these data demonstrate that the 58-kD protein
was Kar5p. In some experiments (e.g., Fig. 7), the Kar5p band was resolved into a doublet. The molecular nature of
the two bands has not been determined. Overexpression
of KAR5 had no detectable adverse phenotype (data not
shown). In addition, both a low molecular mass protein
(Fig. 5) and a slightly higher molecular mass protein (Fig.
7) were sometimes detected with the antibody. Since these
proteins were observed in the kar5- strain, they are not
derived from Kar5p.
To determine if Kar5p was enriched in nuclear membranes, nuclei were isolated by the method of Aris and
Blobel (1991). The enriched nuclear proteins were electrophoretically separated by SDS-PAGE and analyzed by
protein blot. Kar5p was found to be highly enriched in the
purified nuclei (Fig. 6). As a control, we also examined the
ER/nuclear envelope protein Kar2p and found it to be enriched in the nuclear fraction.
The sequence data suggested that Kar5p was an integral
membrane protein. After low-speed (10,000 g) centrifugation of crude yeast lysates, all detectable Kar5p was found
in the pellet fraction (not shown). These results were consistent with Kar5p being a nuclear protein and suggested
that Kar5p might be a membrane protein. To determine
directly whether Kar5p was a membrane protein, we attempted to extract Kar5p from whole nuclei using a variety of treatments (Fig. 7). For controls, we also followed
the extraction properties of Sec72p, a peripheral nuclear
envelope protein; Kar2p, a lumenal protein; and Sec61p,
an integral membrane protein. As previously reported,
Sec72p was not released under conditions of high salt (1 M
NaCl) or high pH (0.1 M Na2CO3, pH 11.0) but was partially released by detergent (1% Triton X-100) (Feldheim and Schekman, 1994). Sec72p was completely released by
the combination of detergent and high salt (1% Triton
X-100 + 0.5 M NaCl). Kar2p was partially released under
most conditions but was extensively released by treatments that disrupt membrane integrity, such as high pH
and detergent. The most extensive release was observed with the combination of detergent and high salt. Sec61p
was slightly released with detergent treatment and released completely in the presence of 0.5 M NaCl (Stirling
et al., 1992
). Kar5p was not extracted by conditions that
released the lumenal membrane proteins nor was it extracted by detergent alone. However, Kar5p was partially solubilized by the presence of detergent and high salt.
Taken together, these results indicate that Kar5p is most
likely an integral membrane protein. The relatively poor
extraction of Kar5p by detergent suggests that Kar5p may
be complexed with other proteins in the nuclear envelope.
To determine the orientation of Kar5p with respect to
the ER membrane/nuclear envelope, the sensitivity of
Kar5p to protease was examined. Proteins exposed on the
surface of the membrane are sensitive to exogenous proteases. In contrast, proteins contained within the lumen
between the inner and outer nuclear membranes are resistant to the addition of proteases. Lumenal proteins would become sensitive to proteases upon addition of detergent
to disrupt the membrane. Sec72p, a peripheral ER/nuclear
envelope protein (Feldheim and Schekman, 1994), was sensitive to proteinase K (Fig. 8) regardless of the presence of
detergent. In contrast, Kar2p, an ER-lumenal protein
(Normington et al., 1989
; Rose et al., 1989
), was resistant
to proteinase K in the absence of detergent but sensitive in
the presence of detergent. Like Kar2p, Kar5p was resistant to protease in the absence of detergent but sensitive in its presence. The detergent-dependent protease sensitivity of Kar5p indicates that the protein was not inherently protease resistant. Furthermore, the fact that Kar5p
was not significantly trimmed by protease in the absence
of detergent suggests that very little if any of the protein is
exposed on the cytoplasmic side of the membrane. This is
consistent with the observation that the putative transmembrane domains are present at the extreme carboxyl terminus of the protein. Taken together, these results demonstrate that Kar5p is a nuclear envelope/ER membrane
protein, anchored in the membrane by one or more hydrophobic transmembrane domains and topologically oriented such that most or all of the hydrophilic portion of the
protein is situated within the ER/nuclear envelope lumen.
Kar5p Is Associated with the Spindle Pole Body during Shmoo Formation
To better define the cellular location of Kar5p, cells were
stained by indirect immunofluorescence using the affinity-purified anti-Kar5p antibodies. Since Kar5p staining was
found to be sensitive to formaldehyde fixation, cells were
either fixed in paraformaldehyde or processed using the
methanol-acetone method of Rout and Kilmartin (1990; see
Materials and Methods). Preadsorbed affinity-purified anti-Kar5p antibody was used for indirect immunofluorescence
on wild-type, kar5-, and Kar5p overexpressing strains.
DAPI staining was used to visualize the nucleus. Kar5p
was detectable at wild-type levels in pheromone-treated
cells fixed in methanol-acetone. However, by this method
the overall cellular morphology was poorly preserved. Paraformaldehyde fixation followed by SDS extraction resulted in better preservation of the cellular morphology, but
Kar5p was then detectable only in pheromone-treated
cells containing KAR5 on a multicopy plasmid. Kar5p was
not detected in vegetative cells or in zygotes by either
method.
In wild-type cells, and in cells in which Kar5p was overexpressed, Kar5p localization was confined to a spot on
the periphery of the nucleus (Figs. 9 and 10). Most cells
had a single large spot of localization, but some cells had
one or more additional smaller spots along the nuclear periphery (71% had one spot, 19% had two spots, and 10%
had three spots, out of 105 cells counted). The minor spots
were largely observed in cells using methanol-acetone fixation and only rarely in cells fixed with paraformaldehyde. The minor spots may therefore result from inadequate fixation.
To identify the location of the Kar5p spot, we performed colocalization experiments using a monoclonal antibody specific for the 90-kD protein, a major protein of
the spindle pole body (SPB). For this experiment, detection of the 90-kD antigen requires use of acetone-methanol fixation. In all cells, a Kar5p spot colocalized with the
90-kD SPB marker (Fig. 9, compare B with C and E with F). In 74% of cells (50 of 68 shmoos counted), the major
Kar5p spot and the 90-kD protein colocalized. In the remaining cells, one of the minor Kar5p spots colocalized
with the 90-kD protein. To demonstrate that Kar5p staining was specific and not due to bleedthrough fluorescence
from the 90-kD staining, the colocalization experiment
was performed on kar5-2 cells. Kar5p was not detected in
kar5-
2 cells (Fig. 9 C). Furthermore, Kar5p/SPB staining
was not observed in the absence of anti-Kar5p antibody
(data not shown).
To examine the location of the Kar5p spot with relation
to the overall cell morphology, paraformaldehyde-fixed
shmoos were stained with the preadsorbed/affinity-purified anti-Kar5p antibodies (Fig. 10). By this method, 75%
of all wild-type shmoos (85/113) stained with the anti-Kar5p antibody. Kar5p was present as a spot or bar on the
nuclear periphery (Fig. 10, middle). Consistent with SPB
localization (Byers and Goetsch, 1974; 1975
), the Kar5p
spot was observed on the face of the nucleus pointed towards the shmoo tip in 77% of wild-type shmoos examined
(57 stained cells). To test the specificity of the anti-Kar5p
staining under these conditions, immunofluorescence was
performed on kar5-
2 shmoos (Fig. 10 B, middle); as before, in kar5-
2 cells, the Kar5p spot was not detected. Thus, by two different fixation and staining techniques,
Kar5p was observed as a spot on the nuclear periphery.
Based upon these localization data, we conclude that Kar5p
is associated with the SPB during mating, where it would
be in position to participate in nuclear membrane fusion
during karyogamy.
KAR5 Is a Mating-induced Gene Required for Nuclear Membrane Fusion
KAR5 is a gene required for the membrane fusion between haploid nuclei during mating in Saccharomyces cerevisiae (Kurihara et al., 1994). In this paper, we report the
cloning and sequencing of the KAR5 gene, the phenotype
of KAR5 gene disruptions, and the subcellular localization
of the Kar5 protein. KAR5 is a nonessential gene encoding
a novel 58.4-kD membrane protein with predicted coiled-coil domains.
The properties of KAR5 and its encoded protein indicate that Kar5p plays a central role in nuclear envelope fusion during karyogamy: (a) Strains bearing a disruption of
the KAR5 gene manifested severe karyogamy defects in
bilateral or "self" matings. As for the original KAR5 mutants (Kurihara et al., 1994), the two nuclei were found
closely apposed but unfused in zygotes formed during the
mating of kar5-
haploids. (b) Expression of KAR5 was
induced upon addition of pheromone consistent with a
specific role in nuclear membrane fusion during karyogamy, and induction was dependent upon the STE12 transcriptional activator. Thus, KAR5 joins a growing list of
genes including FUS1, FUS2, MFA1, CIK1, KAR3, and
KAR4 that are downstream targets of the mating-specific
MAP kinase-activated transcriptional regulon (for recent
reviews see Sprague and Thorner, 1992
; Rose, 1996
). Indeed, KAR5 has been identified recently as FIG3 in a
screen for genes that are specifically induced by mating pheromone (Erdman, S., and M. Snyder, personal communication) (c) Kar5p was found to be a nuclear membrane
protein. Thus, Kar5p is associated with the membrane
whose fusion it helps mediate. (d) The dependence of nuclear membrane fusion on Kar5p has been recapitulated in
vitro. It was previously shown that the original kar5 mutants are defective for membrane fusion in vitro (Kurihara et al., 1994
). Recently, it has been shown that, like the
original kar5 mutants, the kar5-
strains are also defective
in the in vitro ER homotypic fusion assay (Latterich, M.,
personal communication). The fusion defect could not be
rescued by the addition of wild-type cytosol, and anti-Kar5p antisera did not inhibit the fusion of wild-type
membranes in vitro (Latterich, M., personal communication). These observations are consistent with our findings
that Kar5p sedimented with membranes and was not
present in cytosolic fractions. Moreover, Kar5p was localized within the ER/nuclear envelope lumen, shielded from
exogenously added proteases and potentially inhibitory antibodies. (e) Lastly, in shmoos Kar5p was localized to
the vicinity of the SPB, the initial site of membrane fusion
between zygotic nuclei. This localization at the SPB in
shmoos was observed before cell fusion or karyogamy began. Based on these results, it is clear that Kar5p plays a
critical function in membrane fusion during karyogamy.
kar5 Is a Bilateral Karyogamy Mutation Affecting a "Nondiffusible" Protein
Mutant alleles of KAR5 were identified in a genetic screen
specifically tailored to identify bilateral karyogamy mutants (Kurihara et al., 1994). The previously isolated kar5-486 and kar5-1162 alleles, as well as kar5-
alleles, only
manifested karyogamy defects when both partners in the
mating were mutant. The bilateral defect of some KAR
mutants may be a consequence of the special conditions
that arise during karyogamy. After cell fusion but before
nuclear fusion, both haploid nuclei briefly coexist in the
same zygotic cytoplasm. In the common milieu of the zygote, gene products are free to diffuse between nuclei. In a
bilateral mutant, the karyogamy defect may not be manifested in matings with wild-type cells either because the
corresponding wild-type factor is free to diffuse to the mutant nucleus, where it can suppress the defect, or because
it is required in only one of the two nuclei to promote nuclear fusion. In contrast, recessive unilateral mutants, like
those represented by some of the initially identified KAR
genes (Conde and Fink, 1976
; Polaina and Conde, 1982
),
may be the result of compartmentalization or membrane
attachment of corresponding gene products. For example,
Kar2p is not free to diffuse between nuclei because it is restricted to the ER lumen (Normington et al., 1989
; Rose et
al., 1989
), and Kar1p is anchored to the spindle pole body
(Vallen et al., 1992
). However, if Kar5p is compartmentalized within the ER lumen, why are KAR5 mutants bilaterally defective? It seems unlikely Kar5p is required in only
one of the two nuclei to promote nuclear fusion because it was required in both nuclei for in vitro homotypic membrane fusion (Kurihara et al., 1994
). It also seems unlikely
that mature Kar5p is diffusible between the nuclei. However, it is likely that KAR5 mRNA and nascent Kar5p are
freely transferred. During karyogamy, Kar5p levels are substantially increased as a result of STE12-mediated transcriptional induction. As a result, in the common milieu of the zygote, the induced KAR5 mRNA from one haploid
nucleus can be translated on the ER associated with either
nucleus, effectively transferring Kar5p to the mutant nucleus. In contrast, neither KAR1 and KAR2 are induced
by mating pheromone. Accordingly, we propose that for
KAR1 and KAR2, the levels of mRNA expressed during mating would be insufficient to deliver enough wild-type
gene product to the mutant nucleus to correct the defect in
unilateral matings.
Possible Functions of KAR5 during Nuclear Membrane Fusion
Assuming that nuclear envelope fusion is similar to other
membrane fusion systems, there must be several steps to
the overall process (for review see White, 1990). First, microtubule-dependent congression would serve to bring the
two nuclei close together. Second, there must be a docking/recognition event in which the fusogen proteins in the
two membranes are brought into close apposition. Third,
the initiation of fusion between the outer membrane leaflets would result in "hemifusion intermediates." Fourth,
the opening of the pore and its dilation would complete
the outer membrane fusion event. The subsequent events
of nuclear envelope fusion are less clear. Two different
models have been proposed that differ in their requirements for additional inner nuclear membrane fusion events
to produce the single diploid nucleus (Rose, 1996
).
In keeping with other membrane fusion reactions, we
presume that the proteins mediating the initial steps of nuclear envelope fusion, docking and initiation, would extend out of the nuclear envelope into the cytoplasm. However, as determined by protease protection, Kar5p was
confined within the lumen of the ER/nuclear envelope, with
no significant exposed cytoplasmic domain. As such, it is
unlikely that Kar5p could directly mediate the recognition, docking, or initiation of membrane fusion. More likely is
that Kar5p is required for the completion of membrane fusion once initial contact between nuclei has been made.
Consistent with this hypothesis is the observation that
electron micrographs of kar5 mutant zygotes showed small
lipid bridges spanning the cytoplasm between the two nuclear envelopes (Kurihara et al., 1994; Beh, 1996
). According to these studies, the two nuclei made several limited
contacts in the mutant zygotes but, as judged by both biochemical and genetic assays, nevertheless failed to complete fusion. In this view, Kar5p may be a factor required
to dilate or expand the initial fusion pore. One possibility
is that Kar5p could directly promote fusion by destabilizing the membrane bilayer from the underside. Such an activity has been demonstrated for amphipathic peptides, which can aid in the conversion of hemifusion intermediates (Melikyan et al., 1997
).
Other aspects of Kar5p's function in karyogamy are suggested by its regulation and localization. After pheromone induction, Kar5p accumulated at the spindle pole body in shmoos. Therefore, Kar5p can localize to the eventual site of membrane fusion before cell or nuclear fusion. Indeed, to date Kar5p is the only protein required for nuclear membrane fusion that is pheromone induced. Furthermore, none of the other proteins required for nuclear membrane fusion that have been subcellularly localized are specifically localized to the spindle pole body. Therefore, Kar5p might regulate nuclear fusion by virtue of its transcriptional regulation and subcellular localization to designate the timing and location of membrane fusion. In this view, Kar5p might also serve as a scaffold for the localization of the other proteins required for fusion to the vicinity of the spindle pole body.
As diagrammed in Fig. 11, the SPB is a multiprotein complex of at least three layers; a dense central plaque is sandwiched between two plaques of lesser density (Winey and
Byers, 1993). In pheromone-arrested cells, one edge of the
central plaque abuts the "half-bridge," a region of darkly
staining nuclear envelope. Nuclear envelope fusion initiates along one edge of the half-bridge and continues
along the SPB such that two SPBs fuse to a form a single
enlarged SPB (Byers and Goetsch, 1975
). A crease marking the edge of fusion can be observed in the zygotic SPB.
Kar5p was clearly localized to the region of the SPB by
immunofluorescence. However, Kar5p was more diffusely
localized than the 90-kD SPB component, suggesting that
Kar5p was associated with but not actually a central component of the SPB. A similar SPB-associated localization
pattern has been described for the Nuf2 protein (Osborne
et al., 1994). Given this pattern, Kar5p may be associated
with the half-bridge and nuclear membrane adjacent to
the SPB. Determination of the precise localization of
Kar5p awaits future studies by immunoelectron microscopy.
Interactions between Membrane Fusion-specific KAR Genes
A number of genes have been reported to be required in
vivo for the membrane fusion step of karyogamy, including JEM1, KAR2, KAR5, KAR7, KAR8, SEC63, SEC71,
and SEC72 (Polaina and Conde, 1982; Kurihara et al., 1994
;
Ng and Walter, 1996
; Nishikawa and Endo, 1997
). All of
the characterized protein components required for nuclear
membrane fusion are depicted in Fig. 11. Several of the
genes were first identified because of their roles in the translocation of proteins into the lumen of the ER or because their cognate proteins interact with the translocation
machinery. SEC63, SEC71, and SEC72 were originally identified in screens for translocation-defective mutants (Toyn
et al., 1988
; Green et al., 1992
). Sec63p is a DnaJ homologue resident in the ER/nuclear envelope (Sadler et al.,
1989
). Both genetic and biochemical experiments demonstrate that Sec63p and Kar2p interact (Brodsky and Schekman, 1993
; Scidmore et al., 1993
). SEC71 was also identified as a high copy suppressor of a mutation in SEC63
(Kurihara and Silver, 1993
) and biochemically as a Sec63p
interacting protein (Feldheim et al., 1993
). KAR7 has recently been discovered to be identical to SEC71 (Brizzio,
V., and M.D. Rose, unpublished data). Sec71p, Sec72p,
and Kar2p copurify with Sec63p as a complex (Brodsky
and Schekman, 1993
). Finally, Jem1p was recently identified as another DnaJ homologue resident in the nuclear
envelope (Nishikawa and Endo, 1997
). The jem1 mutant
results in a strong defect in nuclear fusion but has little effect on vegetative growth.
The requirement of several of the nuclear fusion proteins for both translocation and nuclear fusion raises the
question of whether they act directly in nuclear fusion or
are required only for the translocation of a protein like
Kar5p into the ER. Several genetic and biochemical results suggest that these proteins play a role in fusion that is
distinct from their role in translocation. First, there exist
mutant alleles of KAR2 that have strong defects in nuclear
fusion that have little if any defects in translocation (Vogel
et al., 1990). Second, Kar2p and Kar7p/Sec71p are required in vitro for homotypic membrane fusion, under
conditions in which protein translocation is not occurring
(Latterich and Schekman, 1994
). Indeed, in this assay temperature-sensitive kar2 and sec71/kar7 mutations cause
temperature-sensitive defects in vitro (Kurihara et al.,
1994
; Latterich and Schekman, 1994
; Brizzio, V., M. Latterich, M.D. Rose, unpublished data). These data suggest
that both Kar2p and Sec71p directly affect nuclear fusion
and are not simply required for the translocation of fusion
proteins into the nuclear envelope/ER. One reasonable
suggestion is that the Kar2p, Sec63p, Sec71p, and Sec72p
are components of a protein complex with a primary role in translocation and a secondary role in membrane fusion
(Ng and Walter, 1996
).
With respect to nuclear fusion, mutations in KAR5 and
KAR7 showed complex genetic behaviors that suggested
that their cognate proteins may interact or be functionally
related. For example, the mutations kar7-1039 and kar5-1162 exhibited unlinked noncomplementation, whereas
kar7-1039 and kar5-468 showed normal complementation
behavior (Kurihara et al., 1994). Furthermore, although
neither kar7 nor kar5 mutants show strong nuclear fusion
defects when crossed to wild-type strains (the bilateral mutant phenotype), they exhibit a strong defect when crossed
against one another (Brizzio, V., and M.D. Rose, unpublished). One explanation for these genetic interactions is that
the functions of Kar5p and Kar7p in nuclear fusion are in
some way interdependent. For example, one attractive hypothesis is that Kar5p and Kar7p/Sec71p are components
of the same nuclear fusion protein complex. Kar5p might
serve to localize and concentrate a subset of Sec63p/Kar2p/
Sec71p/Sec72p complexes to the vicinity of the SPB where
nuclear fusion will take place.
The location, regulation and mutant phenotypes of Kar5p demonstrate that it is a unique protein required specifically for nuclear membrane fusion during mating. Future directions for research will include elucidating its interactions with other components in the ER/nuclear envelope as well as its specific role in membrane fusion during mating and mitosis.
Received for publication 16 May 1997 and in revised form 3 October 1997.
Thanks to Jasper Rine, Sue Biggins, and Nancy Hawkins for critical reading of this manuscript and to Jim Broach, Gerry Waters, Scott Ederman, Mike Snyder, and Martin Latterich for reagents, useful comments, and communicating unpublished results. We also thank Don Huddler and especially Laurie Jo Kurihara for invaluable suggestions and comments.
DAPI, 4,6-diamidino-2-phenylindole;
GST, glutathione-S-transferase;
PRE, pheromone response element;
SC, synthetic complete;
SPB, spindle pole body.
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