Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103
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Abstract |
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The mechanisms by which kinesin-related
proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many
microtubule functions in yeast. Some of these functions
require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J.
Cell Biol. 124:507-519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein
with sequence and structural similarity to Cik1p. The
Vik1 protein is detected in vegetatively growing cells
but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from
that of the Kar3p-Cik1p complex. Vik1p localizes to the
spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle
poles during vegetative growth requires the presence of
Vik1p, but not Cik1p. Phenotypic analysis suggests that
Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to
the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1 mutants. The
vik1
and kar3
mutations, but not cik1
, partially suppresses the temperature-sensitive growth defect of
strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate
that Kar3p forms functionally distinct complexes with
Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.
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Introduction |
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MICROTUBULES are a component of all eukaryotic
cytoskeletons and are essential for many diverse
cellular and intracellular movements, such as
flagellar motility, organelle positioning, vesicle trafficking,
mitotic spindle orientation, and chromosome segregation.
Two families of chemomechanical motor proteins, dynein and kinesin-related proteins, are crucial for these microtubule functions (Vale et al., 1985; Schnapp and Reese, 1989
;
Steuer et al., 1990
; reviewed by Vale and Fletterick, 1997
;
Hirokawa, 1998
). These proteins possess highly conserved
motor domains responsible for movement along microtubules (Yang et al., 1990
). The nonmotor portion of these
proteins links the motor domain to a vast array of cargoes
or localizes the motor to various subcellular sites (Yang
et al., 1989
; Afshar et al., 1995
; Wittmann et al., 1998
).
The family of proteins sharing a region of homology
with the motor domain of conventional kinesin-heavy chain
(KHC)1 has grown enormously in recent years (reviewed
by Hirokawa, 1996; Moore and Endow, 1996
). These kinesin-related proteins (KRPs) have been identified in all eukaryotes from yeast to humans. KRPs vary in directionality and the position of their motor domains. Most KRPs,
as well as conventional kinesin, have NH2-terminal motor
domains and travel toward the more dynamic plus ends of microtubules. Others have internally located motor domains and may also have plus-end polarity. Another subfamily of KRPs have COOH-terminal motors and travel
toward the less dynamic minus ends of microtubules, the
end generally associated with microtubule-organizing centers. Whereas KRPs share a high degree of sequence similarity within their motor domains, the remainder of their
primary sequences are highly divergent. These variable regions are thought to interact with different proteins to mediate the functional specificity of the motor (Page et al.,
1994
). Recent studies have unveiled many details about
the molecular structure and function of kinesin-like motor
domains (Hirose et al., 1995
, 1996
; Arnal et al., 1996
; Kull
et al., 1996
; Sablin et al., 1996
; Gulick et al., 1998
), but far
less is known about how these proteins are regulated and what other components are involved in their function.
Aside from the light chains of conventional KHC, few
other kinesin-associated proteins (KAPs) have been identified. In many organisms, KHCs associate with various
isoforms of kinesin-light chains (KLCs) to form complexes
involved in transport of organelle and vesicle populations
(Cyr et al., 1991; Beushausen et al., 1993
; Gauger and
Goldstein, 1993
; Wedaman et al., 1993
; Khodjakov et al.,
1998
; Rahman et al., 1998
). Additionally, KHC has been shown to tightly associate with both a KLC kinase and a
KLC phosphatase that putatively regulate motor function
(McIlvain et al., 1994
; Lindesmith et al., 1997
). Two KAPs
that interact with specific KRPs have also been identified.
The KIF3 heterodimeric class of KRPs, also known as
kinesin-II, interact with a single KAP to form a heterotrimeric complex (Cole et al., 1993
, 1998
; Scholey, 1996
; Wedaman et al., 1996
; Yamazaki et al., 1996
). Finally, a Saccharomyces cerevisiae KRP, Kar3p, has been shown to
associate with the Cik1 protein, which is essential for
Kar3p localization and function during mating (Page et al.,
1994
).
The Kar3 protein is one of six KRPs encoded by the
S. cerevisiae genome (Meluh and Rose, 1990). KAR3 encodes a protein containing a kinesin-motor domain at its
COOH terminus (Meluh and Rose, 1990
). The Kar3p-motor domain possesses minus-end directionality and microtubule-depolymerizing activity in vitro (Endow et al.,
1994
). In addition to an essential role in nuclear fusion
during mating, or karyogamy, Kar3p has been implicated
in several microtubule functions during the vegetative cell
cycle. These putative functions include spindle assembly,
mitotic chromosome segregation, microtubule depolymerization, kinetochore-motor activity, spindle positioning, and as a force opposing the action of other KRPs (Meluh
and Rose, 1990
; Roof et al., 1991
; Saunders and Hoyt,
1992
; Hoyt et al., 1993
; Endow et al., 1994
; Middleton and
Carbon, 1994
; Cottingham and Hoyt, 1997
; DeZwaan et
al., 1997
; Saunders et al., 1997a
,b; Huyett et al., 1998
). This
presents an interesting problem: how can one motor protein perform such a diverse array of functions within a single cell?
The role of Cik1p during mating is to target Kar3p to
cytoplasmic microtubules (Meluh and Rose, 1990; Page
et al., 1994
). Kar3p and Cik1p are interdependent for
their localization to the SPBs and cytoplasmic microtubules of cells treated with mating pheromone (Page et
al., 1994
). Expression of KAR3 and CIK1 is increased
upon exposure to pheromone, but both genes are also expressed during vegetative growth (Meluh and Rose,
1990
; Page and Snyder, 1992
; Kurihara et al., 1996
).
Cik1p is also involved in a subset of Kar3p's vegetative
functions. kar3
and cik1
mutants share several vegetative phenotypes, including a growth defect at 37°C,
enhanced cytoplasmic microtubules, very short mitotic spindles, and an accumulation of large budded cells indicative of a mitotic cell-cycle checkpoint delay (Meluh
and Rose, 1990
; Page and Snyder, 1992
; Page et al.,
1994
). They also share genetic interactions with several
genes (Manning et al., 1997
). Furthermore, Cik1p requires
Kar3p for its mitotic spindle localization (Page et al., 1994
),
and the two proteins coimmunoprecipitate from vegetative cell lysates (Barrett, J.G., B.D. Manning, and M. Snyder,
unpublished data).
However, unlike during mating, Kar3p does not require
Cik1p for its localization to the spindle poles in mitosis
(Page et al., 1994; this study). This suggests that Kar3p has
some Cik1p-independent functions. Genetic studies support this hypothesis. Kar3p is believed to oppose the force
generated by two other S. cerevisiae KRPs, Cin8p and
Kip1p, which are involved in spindle pole separation both
during spindle assembly and during anaphase B spindle
elongation (Hoyt et al., 1992
, 1993
; Roof et al., 1992
; Saunders and Hoyt, 1992
; Saunders et al., 1995
). Disruption of
KAR3 function partially rescues the temperature-sensitive
growth defect and spindle collapse phenotype of cin8ts
kip1
mutants (Saunders and Hoyt, 1992
; Hoyt et al.,
1993
). In contrast, disruption of CIK1 does not rescue this
mutant (Page et al., 1994
; this study). Together, these results suggest that Kar3p may perform some of its vegetative functions alone or in association with a different KAP.
In this study we describe a Cik1p-homologous protein in
S. cerevisiae that acts as a second KAP for Kar3p. We
demonstrate that this protein, Vik1p (vegetative interaction with Kar3p), is present in vegetatively growing cells
but absent from mating-pheromone treated cells. Vik1p
forms a complex with Kar3p that is distinct from that
between Kar3p and Cik1p. Furthermore, we show that
Kar3p and Vik1p are interdependent for their concentration at the poles of the mitotic spindle. Phenotypic and
genetic comparisons of cik1 and vik1
mutants demonstrate that Cik1p and Vik1p are likely to mediate distinct
subsets of Kar3p functions. Our data suggest that Cik1p
and Vik1p regulate Kar3p function, at least in part, by targeting the motor to various sites of action within the cell. This is the first example of two distinct associated proteins differentially regulating a single KRP.
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Materials and Methods |
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Strains, Media, and Standard Methods
S. cerevisiae strains used in this study are listed in Table I. Yeast growth
media, molecular biological techniques, and genetic manipulations were
as described previously (Sambrook et al., 1989; Guthrie and Fink, 1991
).
Yeast transformation procedures were performed using the lithium acetate method (Ito et al., 1983
). Where indicated, rich medium, consisting of
yeast extract, peptone, and dextrose, was supplemented with benomyl
(DuPont), dissolved in DMSO, to a final concentration of 10, 20, or 30 µg/ml.
Sensitivity of wild-type yeast strains to these concentrations of benomyl varied between preparations of benomyl containing agar plates and,
hence, growth comparisons were always performed on the same plate.
Sensitivity also varies dramatically between growth temperatures (i.e., at
23°C strains are much more benomyl sensitive than at 30°C).
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Construction of VIK1::3XHA Strain
A strain containing the VIK1::3XHA allele (Y1744) was constructed by
the PCR-epitope tagging method described previously (Schneider et al.,
1995). The primers 5'-TATTAACGATTTCAGAAGAAGTTCAAACACAACTTTGTAAAAGAAAGAAAAAGCTCACTAGGGAACA-AAAAGCTGG-3' and 5'-CTTATTTGTTTCATATCTAAATGGCTGTG TTAAGAAAGACGATAATG TGACCGAGC TTAC TATAGG-GCGAATTGG-3' were used in a PCR reaction with pMPY-3XHA as the
template. The resulting 1.5-kb PCR product contains the URA3 gene
flanked by direct repeats encoding three copies of the hemagglutinin
(HA) epitope and contains 59 bp of sequence from the 3' end of the VIK1
gene at one end and 59 bp of sequence downstream of, and including, the
VIK1 translation termination codon at the other end. This fragment was
used to transform yeast strain Y1731, and transformants were selected on
synthetic complete medium lacking uracil. Correct integration into the 3'
region of the VIK1 locus was confirmed by PCR analysis with primers to
sequences flanking the site of insertion. Transformants with a correct
3XHA-URA3-3XHA integration were then incubated on plates containing 5-fluoroorotic acid to select for loss of the URA3 marker by recombination between the repeated 3XHA regions. The resulting VIK1::3XHA
allele was confirmed by PCR and immunoblot analysis. This allele does
not display any vik1
phenotypes; growth of VIK1::3XHA strains in the
presence of benomyl is identical to wild-type strains, and the temperature-sensitive growth defect of cik1
mutants is the same in a VIK1 or VIK1::
3XHA background.
Immunoprecipitations and Immunoblot Analysis
Cells were grown in rich liquid medium to mid-logarithmic phase (OD600 = 0.5-0.8), and a total of 10 OD600 units of cells were collected by centrifugation, washed, and resuspended in 100 µl lysis buffer (1 M NaCl, 10 mM
EDTA, 2 mM EGTA, 5% glycerol, 40 mM Tris-HCl, pH 7.5, containing
1 µl yeast protease inhibitor cocktail (Sigma Chemical Co.) and 200 µM
PMSF). When indicated, cells were first washed twice with fresh medium,
resuspended in medium containing 5 µg/ml -factor mating pheromone
(Sigma Chemical Co.), and incubated for 2 h before harvesting. Cell lysates were prepared in Eppendorf tubes using zirconia/silica beads (Biospec Products) with 40-s pulses of vortexing separated by incubations on
ice; this procedure was repeated 6-8 times. Lysates were then centrifuged
for 10 min at 6,500 g, and a 10-µl aliquot was removed for immunoblot analysis.
For immunoprecipitations, the remaining cell lysate and beads were
washed with 500 µl lysis buffer containing detergents (1% NP-40, 0.5%
sodium deoxycholate, and 0.1% SDS) without NaCl for 20 min on a roller
drum at 4°C. Lysates were then cleared of unlysed cells and cell debris by
centrifugation for 10 min at 6,500 g and ~500 µl of supernatant was transferred to a new tube. The 500 µl lysate was brought to a 1-ml vol with the
same lysis buffer plus detergents (100 mM final NaCl concentration). The
cell lysate was then precleared for 1 h by incubation with 20 µl of a 1:1 slurry of protein A/G-agarose (Pierce) and TBS (150 mM NaCl, 50 mM
Tris-HCl, pH 8.0). Immunoprecipitations were performed by incubation of cell lysates with either 2 µl mouse monoclonal anti-HA antibodies (12CA5 from BABCO), 10 µl rabbit polyclonal anti-Cik1p antibodies (Page and Snyder, 1992), or 5 µl rabbit polyclonal anti-Kar3p antiserum
(gift of L.L. Satterwhite, P.B. Meluh, and M.D. Rose, Princeton University, Princeton, NJ; Meluh and Rose, 1990
; Page et al., 1994
) for 2 h. 20 µl
protein A/G-agarose was then added and incubated for 1 h before collection by centrifugation at 2,000 g for 1 min. The protein A/G-agarose antibody complexes were then washed twice with 1 ml TBS containing detergents and protease inhibitors (1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS, 1 µl Sigma yeast protease inhibitor cocktail, 200 mM PMSF)
and once with 1 ml TBS plus protease inhibitors. The final pellet was resuspended in 30 µl Laemmli sample buffer (Laemmli, 1970
).
Proteins from cell lysates and immunoprecipitations were denatured by
incubation at 90°C for 10 min before electrophoretic separation in either
8% or 10% SDS-polyacrylamide gels. For attempts to detect Vik1p-3XHA in mating-pheromone treated cells, a threefold larger volume of
cell lysate was loaded than for vegetative cell lysates (data not shown).
Proteins were then transferred to Immobilon-P membranes (Millipore)
for immunoblot analysis with either mouse monoclonal anti-HA antibodies (12CA5 from BABCO), rabbit polyclonal anti-Cik1p antibodies (Page
and Snyder, 1992), or a crude IgG fraction of the rabbit polyclonal anti-Kar3p antiserum (Meluh and Rose, 1990
; Page et al., 1994
). Reactive protein bands were then detected with alkaline phosphatase-conjugated
secondary antibodies (Amersham) and the CDP Star detection reagent
(Boehringer Mannheim). Overexposure of all blots failed to detect Vik1p-3XHA in cell lysates from mating-pheromone treated cells.
Fluorescence Microscopy
Indirect immunofluorescence was performed as described previously
(Gehrung and Snyder, 1990; Pringle et al., 1991
). Due to sensitivity of the
HA-epitope to formaldehyde fixation, mid-logarithmic phase cells were
fixed with 3.7% formaldehyde for only 15 min. They were then washed
twice with solution A (1.2 M sorbitol, 50 mM potassium phosphate buffer,
pH 6.8), and spheroplasts were prepared by incubating cells in solution A
containing 5 µg/ml Zymolyase 100T, 0.03% glusulase, and 0.2% 2-mercaptoethanol at 37°C for 15 to 30 min. Spheroplasted cells were then
washed and resuspended in solution A and placed onto poly(L-lysine)-
coated slides. Vik1p-3XHA and Kar3p-HAT were detected by incubation
overnight at 4°C with preabsorbed mouse monoclonal anti-HA primary
antibodies (16B12 from BABCO) diluted in PBS/BSA. Bound mouse
anti-HA antibodies were then detected by incubation for 90 min at room
temperature with preabsorbed CY3-conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratory, Inc.). Microtubules were detected by incubation with rabbit anti-yeast
-tubulin
(Tub2p) primary antibodies (gift of F. Solomon, Massachusetts Institute
of Technology; Bond et al., 1986
) followed by incubation with FITC-conjugated goat anti-rabbit secondary antibodies. After both primary and secondary antibody incubations, slides were washed twice with PBS/BSA
and twice with PBS/BSA plus 0.1% NP-40. Finally, slides were mounted
in 70% glycerol, 2% n-propyl gallate, and 0.25 µg/ml Hoechst 33258 to
preserve the preparation and stain DNA for localization of nuclei.
Photographs of representative cells stained with anti-HA antibodies, anti-Tub2p antibodies, and/or Hoechst 33258 were taken, and composite figures were produced and processed using Adobe Photoshop version 3.0 (Adobe Systems, Inc.). Processing procedures were identical for each photo of a particular staining method within a composite figure.
Construction of KAR3::HAT Strains
A strain containing the KAR3::HAT allele was constructed using a transposon insertion technique described previously (Ross-MacDonald et al.,
1997). The entire KAR3 coding region was cloned into the vector pHSS6
and subjected to transposon mutagenesis in Escherichia coli as described
(Ross-MacDonald et al., 1997
). Plasmid DNA was prepared from selected
strains and digested with NotI, producing a fragment containing the mTn-3xHA/lacZ transposon inserted randomly into the KAR3 gene. These
fragments were then used to transform yeast strain Y1869, using the
URA3 marker encoded by the transposon for selection. Transformants
with in-frame transposon insertions into the KAR3 genomic locus were selected by detection of
-galactosidase activity as described (Burns et al.,
1994
). Cre recombinase-mediated excision, leaving only the 93 amino acid
HA/transposon tag (HAT) inserted, was induced by growth on galactose
(pB227 contains cre under control of a galactose-inducible promoter). Recombinants were selected on 5-fluoroorotic acid. Strains with in-frame
HAT insertions were then tested for kar3
mutant phenotypes, such as
defects in karyogamy and temperature sensitivity. PCR and sequence analysis was performed on DNA from fully complementing strains in order to determine the site of insertion within the KAR3 gene.
Strain Y1870 contains a KAR3 allele with a HAT insertion after the
codon for S68, and does not exhibit any kar3 phenotypes. Y1870 was then
crossed with Y1864 to yield Y1871. Y1871 was then sporulated and a
MAT
KAR3::HAT cik1
::LEU2 segregant (Y1751) was isolated. The
VIK1 gene was disrupted with the HIS3 marker in Y1870 yielding Y1752 (see below). Y1752 was then crossed with Y1751 yielding Y1753. Y1753
haploid segregants were used to analyze Kar3p-HAT localization in wild-type, cik1
, vik1
, and cik1
vik1
strains.
Cloning of VIK1 and Disruption of VIK1, CIK1, and KAR3
Sequence of the VIK1 gene was acquired from the Saccharomyces Genome Database (ORF designation YPL253c; Stanford University). A
2,920-bp region, from the MscI site 5' of the predicted VIK1 translation
start site to the SpeI site 3' of the VIK1 translation termination site, was
PCR amplified from yeast genomic DNA and cloned into pBluescript SK
(Stratagene), replacing its EcoRV-SpeI fragment (pSK-VIK1). The SalI-SacII fragment from this plasmid, containing the VIK1 gene and flanking
sequences, was cloned into the SalI-SacII site of the CEN plasmid pRS316
(Sikorski and Hieter, 1989). This plasmid was linearized by deleting the
NruI-AflII fragment containing the entire VIK1 ORF, then gap repaired
in a wild-type yeast strain (Guthrie and Fink, 1991
). This CEN plasmid encoding wild-type VIK1 was used for subsequent phenotypic analysis.
VIK1 disruption constructs were made by replacing the NruI-AflII
fragment of pSK-VIK1 with the SmaI fragment from either pJA50 (HIS3-Kmr) or pJA53 (URA3-Kmr) (Elledge and Davis, 1988). SalI-XbaI fragments from these plasmids, containing either the HIS3 or URA3 selectable markers flanked by the 5' and 3' noncoding regions of VIK1, were
used to transform yeast strains Y270, Y818, and Y1870 yielding vik1
strains Y1733, Y1748, and Y1752, respectively.
CIK1 and KAR3 disruptions were made as previously described (Page
and Snyder, 1992; Page et al., 1994
). The cik1-
3::LEU2 construct was
used to transform strains Y817 and Y1744 yielding cik1
strains Y1758
and Y1745, respectively. The kar3-
4::URA3 construct was used to transform strains Y818 and Y1744 yielding kar3
strains Y1759 and Y1750, respectively.
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Results |
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Vik1p: A Cik1p Homologue Present in Vegetative Cells but Not in Cells Treated with Mating Pheromone
A database homology search with the Cik1p amino acid
sequence identified an open reading frame (ORF) on chromosome XVI of the S. cerevisiae genome (BLAST search
of predicted translation products of S. cerevisiae ORFs;
Saccharomyces Genome Database ORF designation
YPL253c) predicted to encode a protein with significant homology to Cik1p. This gene was named VIK1, for vegetative interaction with Kar3p (see below). VIK1 is predicted to encode a protein of 647 amino acids, with an
overall sequence identity of 24%, and similarity of 37%, to
Cik1p. The Vik1p sequence also shares structural similarity to Cik1p. It contains a predicted -helical coiled-coil
domain (amino acids 80-385) of the same length and location as that found in Cik1p (amino acids 81-388; Page and Snyder, 1992
; Fig. 1; predicted with COILS version 2.2;
Lupas et al., 1991
; Lupas, 1996
); both proteins contain a
short break of 40 amino acids in their coiled-coil regions.
This break and an 80 residue NH2-terminal globular domain are the most divergent regions of the two proteins
(16% identity). One notable difference in the amino terminal domain is that Vik1p lacks a recognizable nuclear
localization signal found in Cik1p (amino acids 24 to 33;
Fig. 1; Barrett, J.G., and M. Snyder, manuscript in preparation). Finally, the COOH-terminal globular domains of
the two proteins share two regions of 134 and 43 amino
acids in length with 25% identity each. Therefore, Vik1p
shares sequence and structural homology to Cik1p which
is not confined to their coiled-coil domains.
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To characterize the Vik1 protein by immunoblot and immunofluorescence analysis, DNA encoding a triple HA-epitope tag was integrated into the COOH-terminal coding region of the VIK1 genomic locus (see Materials and Methods). The resulting VIK1::3XHA fusion allele fully complements VIK1 function (see Materials and Methods). Immunoblot analysis using anti-HA monoclonal antibodies detects a protein of 92-kD in cell lysates and anti-HA immunoprecipitations from VIK1::3XHA strains (Fig. 2 A). This molecular mass is close to the predicted 76-kD of Vik1p plus the triple-HA epitope tag. This fusion protein is not detected in cell lysates or anti-HA immunoprecipitations from VIK1 untagged strains (Fig. 2 A). Therefore, the VIK1::3XHA allele is expressed during vegetative growth and produces a functional protein of the expected size.
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The Vik1p-3XHA protein is not detected in cell lysates
or anti-HA immunoprecipitations from cultures first exposed to the -factor mating pheromone (Fig. 2 A; see
Materials and Methods). In contrast, Cik1p greatly increases in abundance upon exposure to
-factor (Fig. 2 B;
Page and Snyder, 1992
). Consistent with this result, the
region upstream of the VIK1 ORF does not contain any
predicted pheromone-response elements (Kronstad et
al., 1987
). However, the pheromone-inducible CIK1 and
KAR3 genes each contain multiple pheromone-response
elements in their 5' noncoding regions (Meluh and Rose,
1990
; Page and Snyder, 1992
). Thus, since Vik1p is not
present during mating-pheromone induced differentiation, it is not likely to have a significant role during mating.
Vik1p Coimmunoprecipitates with Kar3p
Cik1p physically associates with Kar3p (Page et al., 1994),
and Vik1p shares sequence and structural similarity to
Cik1p; we therefore tested whether Vik1p could also interact with Kar3p. Cell lysates were prepared from wild-type,
kar3
, and cik1
strains expressing either the VIK1::
3XHA allele or untagged VIK1. Proteins from these cell
lysates were separated by SDS-PAGE (Fig. 3 A) or first
immunoprecipitated with anti-Kar3p polyclonal antibodies (Fig. 3 B; Meluh and Rose, 1990
; Page et al., 1994
).
Immunoblot analysis using anti-HA antibodies detects
Vik1p-3XHA in cell lysates from wild-type, kar3
, and
cik1
strains containing the VIK1::3XHA allele but not in
untagged strains (Fig. 3 A). Therefore, Vik1p stability is
not affected by the absence of either Kar3p or Cik1p. Anti-HA immunoblots of proteins immunoprecipitated
with anti-Kar3p antibodies detects Vik1p-3XHA from a
wild-type VIK1::3XHA strain but not from a kar3
VIK1::
3XHA strain or an untagged strain (Fig. 3 B). Therefore,
immunoprecipitation data indicate that Kar3p and Vik1p
physically interact. Furthermore, the Kar3p-Vik1p complex is quite stable, as cell lysates were prepared in a 1 M
NaCl solution (see Materials and Methods).
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Vik1p-3XHA is also detected in anti-Kar3p immunoprecipitations from a cik1 VIK1::3XHA strain (Fig. 3 B),
indicating that Cik1p is not required for this interaction
between Kar3p and Vik1p. Furthermore, an association
between Vik1p and Cik1p is not detected by immunoprecipitation experiments. Vik1p-3XHA is not precipitated from VIK1::3XHA cell lysates by anti-Cik1p polyclonal
antibodies (Fig. 2 A; Page and Snyder, 1992
; Page et al.,
1994
), and, reciprocally, Cik1p is not precipitated from
these cell lysates with anti-HA antibodies (Fig. 2 B). Protein preparations from vegetatively growing cells or
-factor-treated cells produce the same results. Thus, Vik1p
and Cik1p do not appear to be part of the same complex. Together, these results suggest that Vik1p and Cik1p interact with Kar3p separately, and, therefore, form two different complexes during vegetative growth.
Vik1p Localizes to the Spindle-Pole Bodies
To determine the subcellular localization of Vik1p, asynchronous cultures of VIK1::3XHA and VIK1 untagged strains were fixed briefly with formaldehyde and analyzed by immunofluorescence using anti-HA and anti-tubulin antibodies (see Materials and Methods). VIK1::3XHA cells display anti-HA staining concentrated at the yeast microtubule-organizing centers, or spindle-pole bodies (SPBs), in all stages of the cell cycle (Fig. 4, B and C). The position of the SPBs correspond to the brightest foci of anti-tubulin staining. Anti-HA staining at the SPBs is not detected in VIK1 untagged cells (Fig. 4 A). Moreover, detection of Vik1p-3XHA at the SPBs does not require costaining with anti-tubulin antibodies; staining of dots at the edges of both preanaphase and anaphase nuclei is still observed in control cells not stained with anti-tubulin antibodies (Fig. 4 C). Vik1p-3XHA staining is brightest in preanaphase cells with short spindles, where localization is clearly concentrated at the spindle poles (pattern observed in 100% of preanaphase spindles; n > 200; Fig. 4 B). Cells in the G1 phase of the cell cycle, scored as unbudded cells with a single SPB, display SPB staining that is more faint and not observed in every cell (staining observed in 60% of G1 cells; n = 100). Finally, cells in early or late anaphase, as scored by elongated spindles and/or elongated nuclei penetrating the bud neck, display faint staining concentrated at the spindle poles (staining of at least one spindle pole is observed in 73% of anaphase cells; n = 100; Fig. 4 C, inset). Anti-HA staining is not detected along the lengths of spindle or cytoplasmic microtubules in VIK1::3XHA cells. Short fixation times due to sensitivity of the HA-epitope to formaldehyde leads to a diminished number of cytoplasmic microtubules. However, in cells that contain cytoplasmic microtubules, only staining at the SPBs is observed; microtubule staining is not evident. These staining patterns are independent of ploidy, and the nature and position of the epitope tag. VIK1::3XHA/VIK1::3XHA homozygous diploids, and cells expressing a VIK1::3Xmyc NH2-terminal fusion allele stained with anti-myc monoclonal antibodies, exhibit identical patterns to those found in VIK1::3XHA haploid cells (data not shown).
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The localization of Vik1p at, or near, the SPBs at various levels throughout the vegetative cell cycle is very similar to that reported for fusion proteins of Kar3p with either
-galactosidase or HA (Page et al., 1994
; Saunders et al.,
1997; see below). However, in pheromone-treated cells,
when Kar3p localizes to the SPB and cytoplasmic microtubules (Meluh and Rose, 1990
; Page et al., 1994
), Vik1p-3XHA staining above background is not detected (data
not shown). This is consistent with immunoblot data demonstrating the absence of Vik1p in cell lysates from
-factor-treated cells (Fig. 2 A), and further suggests that Vik1p
functions in vegetative cells, but not mating cells.
Spindle-Pole Body Localization of Vik1p is Kar3p Dependent
Since Kar3p and Vik1p physically associate and have similar localization patterns, we investigated whether the
localization of Vik1p to the SPBs was dependent on the
presence of Kar3p. Therefore, Vik1p-3XHA localization
was analyzed by anti-HA immunofluorescence in kar3
cells. Whereas Vik1p-3XHA concentrates at the SPBs in wild-type cells, it does not localize to the SPBs at any stage of the cell cycle in the kar3
mutant. Instead, the protein
is present in cytoplasmic patches that appear to be excluded from the nucleus (SPB staining was not detected in
any cells; n > 300; a representative preanaphase cell is
shown in Fig. 5 B). This pattern is also observed with anti-myc staining in kar3
cells containing the VIK1::3Xmyc
allele (data not shown). These cytoplasmic patches are not
detected in kar3
VIK1 untagged strains (Fig. 5 A). SPB
localization of Vik1p-3XHA can be restored by introducing a CEN plasmid containing KAR3 into the kar3
VIK1::
3XHA strain (Fig. 5 C). This plasmid also restores normal
spindle length, since disruption of KAR3 results in accumulation of cells with short spindles compared with wild-type cells (Meluh and Rose, 1990
). Therefore, the Kar3p-Vik1p association is required for localization of Vik1p to
the SPB.
|
Cik1p also localizes to the SPB in a Kar3p-dependent
manner (Page et al., 1994), and, like kar3
mutants, cik1
mutants accumulate cells with short mitotic spindles (Page
and Snyder, 1992
). Therefore, in order to determine if
Cik1p was involved in localizing Vik1p to the SPB or if the
short spindle phenotype was responsible for Vik1p mislocalization in kar3
mutants, we analyzed Vik1p-3XHA localization in a cik1
mutant. In contrast to kar3
cells,
cik1
cells display Vik1p-3XHA staining concentrated at
the SPBs during all stages of the cell cycle, as observed in
wild-type cells. Furthermore, HA staining is brightest at
the poles of the short preanaphase spindles present in
cik1
cells (a representative cell is shown in Fig. 5 D), indicating that the short spindle phenotype alone is not the
cause of mislocalization of Vik1p. Therefore, these immunolocalization data support the immunoprecipitation experiments; Vik1p does not appear to be part of the same
complex as Cik1p. Furthermore, Vik1p localization to the
SPBs does not require Cik1p function.
Proper Spindle-Pole Body Localization of Kar3p in Vegetative Cells Is Vik1p Dependent
Cik1p is required to localize Kar3p to the SPB and cytoplasmic microtubules during mating but is not required for
Kar3p localization to the mitotic spindle poles (Page et al.,
1994). This suggests that in vegetative cells, Kar3p can either target to the SPBs by itself or interacts with another
protein that is responsible for its concentration at the
SPBs. Based on its localization and association with Kar3p,
Vik1p could fulfill this function. Therefore, Kar3p was
localized in the absence of Vik1p, Cik1p, and both Vik1p and Cik1p.
The subcellular localization pattern of Kar3p has been
determined previously using fusion proteins that do not
fully complement all of the kar3 phenotypes (Meluh and
Rose, 1990
; Page et al., 1994
; Saunders et al., 1997). To create a fully complementing HA epitope-tagged version of
Kar3p, we used a transposon insertion technique described
previously (Ross-MacDonald et al., 1997
; see Materials
and Methods). Using this technique we created several strains containing in-frame insertions of the HAT into random regions of the KAR3 genomic locus. We then tested
these strains for several kar3
phenotypes, such as defects
in karyogamy and meiosis, slow growth, temperature-sensitive growth, enhanced cytoplasmic microtubules, and the
ability to properly localize Vik1p-3Xmyc to the SPBs (Meluh and Rose, 1990
; Page et al., 1994
; Bascom-Slack and
Dawson, 1997
; Saunders et al., 1997a
). Fully complementing alleles were sequenced and found to lie within the region encoding the NH2-terminal globular domain of Kar3p
(data not shown). A strain containing one of these KAR3::
HAT alleles (encoding Kar3p with the 93-amino acid
HAT insertion at S68) was used for immunofluorescence
analysis with anti-HA antibodies.
Localization of Kar3p-HAT was examined in wild-type,
cik1, vik1
, and cik1
vik1
strains. Asynchronous
cultures of haploid segregants from a cik1
/CIK1 vik1
/
VIK1 KAR3::HAT/KAR3::HAT diploid strain were fixed
and prepared for immunofluorescence with anti-HA and
anti-tubulin antibodies (see Materials and Methods). KAR3:: HAT wild-type cells, in all stages of the cell cycle, display anti-HA staining concentrated at the SPB region, as well
as faint patches confined to the nucleus (as determined by
colocalization with anti-tubulin and Hoechst staining, respectively). These fluorescence patterns are not observed
in KAR3 untagged strains, in which background staining is
restricted to the cytoplasm (Fig. 6 A). Consistent with previous Kar3p localization studies (Page et al., 1994
; Saunders et al., 1997), Kar3p-HAT staining is brightest at the
poles of preanaphase spindles (Fig. 6 B; Table II). Most
cells in G1 (80%, 100 G1 cells counted) and anaphase
(86%, 100 anaphase cells counted) also display Kar3p-HAT localization at the SPBs, but this staining is fainter
than that observed in preanaphase cells (data not shown). In contrast to previous studies, Kar3p-HAT localization
along spindle microtubules is not detected in any stage of
the cell cycle. When cytoplasmic microtubules are observed, in wild-type cells or the mutant cells described below, Kar3p-HAT localization is not detected along their
lengths. Hence, Kar3p is most abundant at the SPBs of
cells throughout the cell cycle.
|
|
The effect of disrupting Vik1p and Cik1p function on
Kar3p-HAT localization was determined. Since wild-type
localization is most obvious in preanaphase cells, we quantified Kar3p-HAT staining patterns in cells from asynchronous cultures at this stage (Table II). Similar to wild-type
strains, KAR3::HAT cik1 cells display anti-HA staining
at the SPBs during all stages of the cell cycle; again, the
brightest staining is observed at the poles of the preanaphase spindle (Fig. 6 C; Table II). However, the nuclear patch staining detected in wild-type cells is qualitatively
diminished in the cik1
mutant. Therefore, as described
previously (Page et al., 1994
), Cik1p is not required to localize Kar3p to the SPBs during the vegetative cell cycle.
In contrast, Kar3p-HAT localization during vegetative
growth is dramatically altered in vik1 cells (Fig 6 D; Table II). Kar3p-HAT is no longer concentrated at the SPBs
in these cells; instead, it predominantly displays a bright
nuclear patch localization in all vik1
cells. Moreover,
staining along the lengths of spindle microtubules is detected in many cells (34% of preanaphase cells; Fig 6 D;
Table II). Some vik1
cells also display faint staining in
the vicinity of the SPBs, but not along spindle microtubules (22% of preanaphase cells; Table II). Therefore, although vik1
cells may retain some Kar3p at the SPBs,
Vik1p is required to concentrate, or restrict, Kar3p localization to the SPBs.
To determine if the spindle and faint SPB localization of
Kar3p detected in vik1 cells is due to Cik1p function, we
localized Kar3p-HAT in a cik1
vik1
double mutant.
Kar3p-HAT localization is even more aberrant in the double mutant, with the nuclear patch staining distributed
more diffusely throughout the nucleus in all cells (representative preanaphase cell shown in Fig. 6 E). Detection of
Kar3p-HAT along the spindle or at the SPBs in these cells
is greatly reduced compared with the vik1
mutant (spindle and faint SPB localization is observed in 7 and 9%, respectively, of cik1
vik1
preanaphase cells; Table II). Introduction of a CEN plasmid encoding VIK1 into either
vik1
or cik1
vik1
mutants completely restores Kar3p-HAT concentration at the SPBs (data not shown).
Therefore, during vegetative growth, Vik1p is primarily responsible for SPB targeting, or retention, of Kar3p.
Cik1p may mediate Kar3p localization to less concentrated sites within the nucleus, such as the spindle and/or
nuclear patches.
CIK1 and VIK1 Disruptions Result in Distinct Phenotypes
Cik1p and Vik1p exhibit differences in their expression
patterns, and deletion of CIK1 or VIK1 results in unique
effects on Kar3p localization. Therefore, the phenotypes
resulting from disruption of VIK1 and CIK1 were compared. Suprisingly, vik1 mutants grow similar to wild-type strains at all temperatures. Using a sectoring assay
described previously (Spencer et al., 1990
), we determined that, unlike cik1
mutants that have a severe chromosome
loss defect (Page and Snyder, 1992
), vik1
mutants display
only a threefold increase in frequency of chromosome loss
per cell division relative to wild-type strains (data not
shown). Furthermore, consistent with its absence in mating-pheromone treated cells, vik1
mutants are not defective in karyogamy, as determined by qualitative mating assays. However, compared with wild-type strains, vik1
mutants are resistant to the microtubule-depolymerizing
drug benomyl (on plates containing 10, 20, or 30 µg/ml
benomyl; 10 µg/ml in Fig. 7 C). The benomyl resistance
phenotype always segregates with the vik1
mutation (11 tetrads analyzed).
|
To analyze whether CIK1 and VIK1 are functionally
redundant, we tested whether expression of VIK1 from
a high copy plasmid could suppress the temperature-sensitive growth defect of a cik1 mutant. cik1
strains
containing either the VIK1 plasmid or vector alone are
equally affected by growth at the restrictive temperature (data not shown). A cik1
mutant was then mated with a
vik1
mutant, and the resulting heterozygous diploid
strain was sporulated. Haploid segregants from tetratype
tetrads were then analyzed (eight tetrads analyzed; results
from a representative tetrad are shown in Fig. 7). Wild-type, cik1
, vik1
, and cik1
vik1
segregants all display
similar growth on rich medium at 23°C (Fig. 7 A). However, cik1
mutants are temperature sensitive for growth
at 37°C (Page and Snyder, 1992
), whereas vik1
mutants
grow like wild-type strains (Fig. 7 B). Suprisingly, cik1
vik1
double mutants grow substantially better at 37°C
than cik1
mutants (Fig. 7 B). This result indicates that
disruption of VIK1 partially suppresses the temperature-sensitive growth defect of cik1
mutants. The temperature-sensitive growth defect can be restored to a cik1
vik1
double mutant by introduction of a CEN plasmid
encoding VIK1 (data not shown). Deletion of VIK1 also
partially suppresses the mitotic delay of cik1
mutants, as
scored by the percentage of large budded cells with a single preanaphase nucleus in logarithmic phase cultures
growing at 30°C (Fig. 7 D). 54% of cells from cik1
cultures exhibit the mitotic delay phenotype, whereas only
36% of cells from cik1
vik1
cultures have this phenotype. Wild-type and vik1
cultures each have 22% large
budded cells. These strains were also analyzed for growth
differences in the presence of benomyl (Fig. 7 C). Unlike
vik1
mutants, which display increased resistance to
benomyl, cik1
and cik1
vik1
mutants are slightly more
sensitive than wild-type strains. Therefore, the phenotypic
differences of cik1
and vik1
mutants, combined with
their different effects on Kar3p localization, suggest that
Cik1p and Vik1p are functionally distinct.
The microtubules of the different mutant strains were
examined in fixed cells from asynchronous or hydroxyurea-arrested cultures by immunofluorescence with anti-tubulin antibodies. Under these conditions, the microtubules of vik1 mutants are indistinguishable from those of
wild-type strains. As described previously, cik1
mutants
have very short spindles compared with wild-type strains and have longer, more abundant cytoplasmic microtubules
(Page and Snyder, 1992
). This phenotype is identical to
that reported for kar3
mutants (Meluh and Rose, 1990
;
Saunders et al., 1997). cik1
vik1
double mutants display
microtubules with no significant difference in length or
number to those of cik1
mutants (data not shown). Therefore, if disruption of VIK1 results in defects in microtubule
structure, these defects are not detected under these conditions. Finally, consistent with the hypothesis that Cik1p
and Vik1p function in complexes with Kar3p, the growth
rate and microtubule phenotypes of kar3
, kar3
cik1
,
kar3
vik1
, and kar3
cik1
vik1
mutants are all very
similar (data not shown).
Loss of Vik1p Function Suppresses the cin8-3
kip1 Mutant
One vegetative function of Kar3p may be to oppose the
action of two other S. cerevisiae KRPs, the redundant
Cin8p and Kip1p motors (Hoyt et al., 1992, 1993
; Saunders
and Hoyt, 1992
; Saunders et al., 1997b
). This Kar3p function has been proposed primarily due to genetic interactions between mutations in the genes encoding these
KRPs. Disruption of Cin8p and Kip1p function, using the conditional cin8-3 kip1
mutant, results in a temperature-sensitive growth defect at 35°C that is partially suppressed
by disruption of Kar3p function (Saunders and Hoyt, 1992
;
Hoyt et al., 1993
; see below). However, disruption of
CIK1, encoding a Kar3p KAP, does not suppress cin8-3
kip1
(Fig. 8; Page et al., 1994
), suggesting that the Kar3p
activity that opposes Cin8p and Kip1p is independent of
Cik1p.
|
Since Vik1p was identified as a second KAP for Kar3p
during vegetative growth, we tested whether disruption of
Vik1p function could suppress the temperature-sensitive
growth defect of the cin8-3 kip1 mutant. VIK1, CIK1,
and KAR3 were deleted individually from the cin8-3 kip1
strain, and the resulting mutants were examined for
growth defects at 23 and 35°C (Fig. 8). As demonstrated
previously, the cin8-3 kip1
kar3
mutant grows significantly better than the original cin8-3 kip1
mutant at the
restrictive temperature of 35°C (Saunders and Hoyt, 1992
;
Hoyt et al., 1993
). Likewise, disruption of VIK1 also partially suppresses the cin8-3 kip1
temperature-sensitive
growth defect, as the cin8-3 kip1
vik1
mutant grows at
35°C. Introduction of a CEN plasmid encoding VIK1 into
the cin8-3 kip1
vik1
mutant restores temperature sensitivity to levels identical to the cin8-3 kip1
mutant (data
not shown). In contrast, growth of the cin8-3 kip1
cik1
mutant is significantly diminished at all temperatures compared with the cin8-3 kip1
mutant, suggesting a possible
added defect in the triple mutant. Therefore, like Kar3p,
Vik1p functions antagonistically to Cin8p and Kip1p. This
result further indicates that Cik1p and Vik1p are involved
in distinct Kar3p functions.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cik1p is a previously described kinesin-associated protein
that interacts with the yeast KRP Kar3p (Page and Snyder,
1992; Page et al., 1994
). We have characterized a Cik1p-homologous protein called Vik1p that is present in vegetatively growing cells but, unlike Cik1p, is not detected in
mating-pheromone treated cells. Coimmunoprecipitation
experiments demonstrate that Vik1p also physically associates with Kar3p and that the Kar3p-Vik1p complex is
separate from that of Kar3p and Cik1p. Therefore, Kar3p
interacts with two different KAPs to form distinct complexes within the same cell.
Kar3p and Vik1p Are Interdependent for Proper Localization to the SPBs of Vegetatively Growing Cells
Vik1p requires Kar3p function for its SPB localization.
This suggests that Kar3p-Vik1p complex formation and,
presumably, the minus-end directed microtubule-motor
activity of Kar3p are required to deliver Vik1p to the SPB.
In the absence of Kar3p, Vik1p mislocalizes to cytoplasmic
patches and is excluded from the nucleus. In contrast,
Cik1p mislocalizes throughout the nucleus in the absence
of Kar3p during vegetative growth (Page et al., 1994). Two
possible models for Vik1p localization can be invoked. First, Vik1p may require association with Kar3p for its nuclear import. In support of this idea, Vik1p does not have
sequences predicting a nuclear localization signal. Moreover, Kar3p can target to the nucleus independent of both
Cik1p and Vik1p (Page et al., 1994
; this study). This nuclear import of Vik1p by association with Kar3p would be
analogous to the nuclear import of the yeast
-tubulin
complex. The import of this complex, and its subsequent binding to the nuclear face of the SPB, requires the nuclear localization signal of one of its components, Spc98p
(Pereira et al., 1998
). Alternatively, the Kar3p-Vik1p complex may be associated primarily with the cytoplasmic face
of the SPB. Therefore, absence of Kar3p would result in
release of Vik1p to the cytoplasm specifically, as observed.
At this point, we cannot distinguish between these two
possibilities, but future studies will address whether the Kar3p-Vik1p complex is on the nuclear, cytoplasmic, or
both faces of the SPB.
We demonstrate that, whereas Cik1p is required to localize Kar3p to the SPBs and cytoplasmic microtubules of
mating pheromone-treated cells (Page et al., 1994), Vik1p
is required for proper concentration of Kar3p at the SPBs
of vegetatively growing cells. In the absence of Vik1p,
Kar3p mislocalizes to nuclear patches, and can be seen along spindle microtubules in many cells. Therefore, Vik1p
is required for proper targeting and/or maintenance of
Kar3p at the SPB. Vik1p might mediate interactions between Kar3p and other proteins that tether the complex
to the SPB. Alternatively, Vik1p could prevent release of
Kar3p from the minus-ends of microtubules, where its motor activity would cause it to accumulate.
In the absence of both Cik1p and Vik1p, Kar3p again
mislocalizes but is more diffuse throughout the nucleus.
This suggests that Cik1p may be partially redundant with
Vik1p for targeting Kar3p to the SPB. However, Cik1p's
primary vegetative function may be to direct localization
of Kar3p to other sites within the nucleus, such as nuclear
patches and the spindle. This is supported by the observation that the faint nuclear patch staining of Kar3p seen in
wild-type cells is diminished in cik1 cells. Detection of
Kar3p at these sites is greatly enhanced in the absence of its Vik1p-mediated SPB localization.
Finally, since some residual SPB staining can be detected in a small percentage of cik1 vik1
cells, Kar3p
may have an inherent KAP-independent ability to localize
to the SPB. This localization is likely to depend on its minus-end directed microtubule motor domain, rather than
its non-motor stalk and tail domains. These latter regions
of Kar3p have been shown previously to be sufficient for
its spindle and SPB localization (Meluh and Rose, 1990
;
Page et al., 1994
). Cik1p and Vik1p presumably target
Kar3p to various sites of action through interactions with
its nonmotor domain.
Kar3p Forms Three Functionally Distinct Complexes during the Yeast Life Cycle
Phenotypic, genetic, and biochemical analysis of Kar3p, by
several different groups (Meluh and Rose, 1990; Roof et al.,
1991
; Saunders and Hoyt, 1992
; Hoyt et al., 1993
; Endow
et al., 1994
; Middleton and Carbon, 1994
; Cottingham and
Hoyt, 1997
; DeZwaan et al., 1997
; Saunders et al., 1997a
;
Huyett et al., 1998
), has strongly suggested that Kar3p is a
multifunctional KRP. An intriguing question in the study
of molecular motors is how can one motor protein perform several different functions within a single cell. Our
results indicate that Kar3p interacts with two related proteins to form three complexes that are involved in distinct microtubule-mediated cellular processes. We believe that
the ability of Kar3p to interact with these associated proteins is crucial to its functional versatility. The exact molecular functions of each of these Kar3p complexes are yet
to be defined. However, based on phenotypic and genetic
analysis, as well as localization studies, our data reveal
some possible general roles for the Kar3p-Cik1p complex
during mating and the Kar3p-Cik1p and Kar3p-Vik1p complexes during mitosis (Fig. 9).
|
The best defined of Kar3p's functions is its role in the
nuclear congression step of karyogamy, during which it associates with Cik1p (Meluh and Rose, 1990; Page et al.,
1994
). This complex localizes to cytoplasmic microtubules
even in the absence of the Kar3p motor domain (Meluh
and Rose, 1990
; Page and Snyder, 1992
; Page et al., 1994
),
indicating that the nonmotor region of the complex also
has microtubule-binding capacity. In the absence of either
of these two proteins, microtubules from the SPBs of opposing mating partners fail to interdigitate (Meluh and
Rose, 1990
; Page et al., 1994
). Together, these results suggest a model in which the Kar3p-Cik1p complex acts as a
cross-linker between antiparallel microtubules emanating
from the SPBs of mating partners. The minus-end directed
microtubule-motor activity of Kar3p can then create the
force that pulls the nuclei together by sliding cross-linked
microtubules past one another (Fig. 9 A).
Despite many studies on the function of Kar3p during
vegetative growth, its role during mitosis remains obscure.
Our identification of two Kar3p-interacting proteins with
distinct Kar3p-related vegetative phenotypes should help
elucidate the exact mitotic functions of this KRP. Disruption of either KAR3 or CIK1 results in similar mitotic phenotypes, including very short spindles indicative of a spindle assembly defect (Meluh and Rose, 1990; Page and
Snyder, 1992
; Saunders et al., 1997a
) and a mitotic delay
mediated by the spindle-assembly checkpoint (Roof et al.,
1991
; Manning, B.D., J.A. Wallace, and M. Snyder, unpublished observation). Unlike during mating, the Kar3p-Cik1p complex is in the nucleus during the mitotic cell
cycle, where it associates with the SPBs and, to a lesser extent, spindle microtubules (Page and Snyder, 1992
; Page
et al., 1994
; Saunders et al., 1997a
). Analogous to its role in
karyogamy, the complex may act within the spindle to
cross-link and slide antiparallel microtubules from opposing SPBs past one another, thereby creating an inward
force on the spindle (Fig. 9 B). This force may generate a
tension important for proper spindle assembly. Alternatively, the Kar3p-Cik1p microtubule cross-linking activity
could be crucial to the organization of a bipolar spindle. This spindle assembly defect would account for the chromosome instability phenotype of cik1
mutants (Page and
Snyder, 1992
). Additionally, it is possible that this complex
could play a more direct role in chromosome segregation,
perhaps as a kinetochore motor (Hyman et al., 1992
; Middleton and Carbon, 1994
).
Kar3p is likely to have a separate mitotic function that
is mediated by interaction with Vik1p at the SPBs. The
benomyl resistance phenotype of vik1 mutants, suggests
that the Kar3p-Vik1p complex may be involved in microtubule depolymerization (Fig. 9 C). The motor domain of
Kar3p has been shown to possess minus-end-specific microtubule-depolymerizing activity in vitro (Endow et al.,
1994
), and has been suggested by phenotypic analysis to
depolymerize microtubules in vivo (Saunders et al., 1997).
Kar3p complexed with Vik1p, specifically, might possess
this activity. Alternatively, Kar3p may require interaction
with Vik1p to prevent release from microtubule minus
ends where it catalyzes microtubule depolymerization. At
this point, it is unclear whether this complex acts on cytoplasmic microtubules, the spindle, or both. Disruption of
VIK1 does not result in any detectable differences in the
microtubule structures of fixed cells compared with wild-type strains. It is possible that accumulation of Kar3p on
the spindle, observed in vik1
mutants, could stabilize
these microtubules and account for the benomyl resistance phenotype.
Based on genetic analysis, Kar3p-Vik1p function is detrimental to mutants lacking the plus-end-directed Cin8p
and Kip1p KRPs. These proteins are members of the
BimC family of KRPs and are believed to act as homotetrameric bipolar motor proteins that generate a SPB separating force during spindle assembly and anaphase B
(Hoyt et al., 1992; Saunders et al., 1995
; Kashina et al.,
1997
; Straight et al., 1998
). Disruption of either KAR3 or
VIK1 can suppress the temperature-sensitive growth defect of the cin8-3 kip1
mutant, suggesting that the Kar3p-Vik1p complex may oppose the function of Cin8p and
Kip1p (Fig. 9 C; Saunders and Hoyt, 1992
; Hoyt et al.,
1993
). Interestingly, a TUB2 mutation that stablilizes microtubules can also suppress this mutant (Saunders et al.,
1997a
), suggesting that a defect in the putative microtubule depolymerizing activity of the Kar3p-Vik1p complex
could be sufficient for suppression of cin8-3 kip1
.
The functional interactions between the two vegetative
Kar3p complexes may be complicated. For example, disruption of VIK1 partially suppresses the temperature-sensitive growth defect and mitotic delay of cik1 mutants.
One interpretation of this result is that the two Kar3p
complexes partially oppose one another. It is also interesting to note that cik1
mutants, in which Kar3p's SPB localization is unperturbed, have much stronger phenotypes (Page and Snyder, 1992
) than those of vik1
mutants, in
which Kar3p is no longer concentrated at the SPBs. Therefore, the most critical of Kar3p functions during vegetative
growth may occur at sites other than the spindle poles. Future studies will address these issues, but it is clear from
our current study that Cik1p and Vik1p are not functionally redundant.
KRP Regulation and Targeting by Kinesin-associated Proteins
In general, the functional specificity of KRPs is determined by their nonmotor domains. Some KRPs can target
to their sites of action independent of associated proteins.
For example, the Drosophila Nod protein, involved in chromosome movements during mitosis, contains a DNA binding motif in its nonmotor domain (Afshar et al., 1995).
However, the targeting of many KRPs will likely be mediated through complex formation with nonmotor subunits
(i.e., KAPs). The highly divergent nature of the nonmotor
domains of KRPs (reviewed by Goldstein, 1993
; Vale
and Fletterick, 1997
) suggest that interacting proteins will
also be diverse in sequence. Nevertheless, we expect that
mechanisms of motor targeting by KAPs will exist that are universal.
The light chains of conventional kinesin are the most
studied of all KAPs. Several different KLCs can exist
within a single cell (Cyr et al., 1991; Beushausen et al.,
1993
; Wedaman et al., 1993
; Rahman et al., 1998
), and
these are thought to control the cargo-binding specificity
of KHCs (Khodjakov et al., 1998
; Liao and Gundersen,
1998
). Therefore, it is possible that different KLCs target
kinesin to distinct membranous organelles and vesicles, and it has been suggested that KLC mediates the interaction between kinesin and membranes (Stenoien and Brady,
1997
). Additionally, KLCs might regulate KHC-microtubule binding and/or motor activity by contacting the motor
domain when kinesin is in a folded confirmation (Hackney
et al., 1992
; Verhey et al., 1998
).
Little is known about the regulation and targeting of
specific KRPs. The tail domain of the Xenopus mitotic
KRP, Xklp2, has recently been shown to require cytoplasmic dynein and a microtubule-associated protein, TPX2,
to localize to spindle poles (Wittmann et al., 1998). However, these proteins are not tightly associated KAPs. Our
results demonstrate that Kar3p localization is regulated
through its interaction with two different KAPs, Cik1p
and Vik1p (Page et al., 1994
; this study). These KAPs control various Kar3p functions, at least in part, by targeting
the motor to discrete sites within the cell. Whether Cik1p
and Vik1p modulate motor activity once Kar3p is at these
sites is not yet known. The study of these proteins should
define regulatory strategies used by other KRPs and help elucidate general elements underlying the functional diversity of KRPs.
![]() |
Footnotes |
---|
Address correspondence to Michael Snyder, Department of Molecular, Cellular, and Developmental Biology, PO Box 208103, Yale University, New Haven, CT 06520-8103. Tel.: (203) 432-6139. Fax: (203)432-6161. E-mail: michael.snyder{at}yale.edu
Received for publication 24 December 1998 and in revised form 18 February 1999.
B.D. Manning and J.G. Barrett were supported by National Institutes
of Health (NIH) training grants and NIH grants GM3649 and GM52197,
and this research was funded by NIH grant GM52197.
We thank C. Horak and J. Vogel for critical comments on the manuscript.
We are grateful to L.L. Satterwhite, P.B. Meluh, and M.D. Rose for providing the anti-Kar3p antibodies, F. Solomon for the anti-Tub2p antibodies, and M.A. Hoyt for the cin8-3 kip1 strain.
![]() |
Abbreviations used in this paper |
---|
HA, hemagglutinin; HAT, HA/transposon tag; KAP, kinesin-associated protein; KHC, kinesin-heavy chain; KLC, kinesin light chain; KRP, kinesin-related protein; ORF, open reading frame; SPB, spindle-pole bodies.
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References |
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