From the Departments of a Biochemistry and h Molecular Biotechnology, University of Washington, Seattle, Washington 98195 and the d Department of Molecular, Cellular and Developmental Biology, b Howard Hughes Medical Institute, and k Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309
Received for publication, November 20, 2000, and in revised form, March 1, 2001
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ABSTRACT |
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The yeast spindle pole body (SPB)
component Spc110p (Nuf1p) undergoes specific serine/threonine
phosphorylation as the mitotic spindle apparatus forms, and this
phosphorylation persists until cells enter anaphase. We demonstrate
that the dual-specificity kinase Mps1p is essential for the
mitosis-specific phosphorylation of Spc110p in vivo and
that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides
generated by proteolytic cleavage were identified and sequenced by mass
spectrometry. Ser60, Thr64, and
Thr68 are the major sites in Spc110p phosphorylated by
Mps1p in vitro, and alanine substitution at these sites
abolishes the mitosis-specific isoform in vivo. This is the
first time that phosphorylation sites of an SPB component have been
determined, and these are the first sites of Mps1p phosphorylation
identified. Alanine substitution for any one of these phosphorylated
residues, in conjunction with an alanine substitution at residue
Ser36, is lethal in combination with alleles of
SPC97, which encodes a component of the Tub4p complex.
Consistent with a specific dysfunction for the alanine substitution
mutations, simultaneous mutation of all four serine/threonine residues
to aspartate does not confer any defect. Sites of Mps1p phosphorylation
and Ser36 are located within the N-terminal globular domain
of Spc110p, which resides at the inner plaque of the SPB and binds the
Tub4p complex.
Centrosomes are microtubule-organizing centers that serve as the
poles of mitotic spindles during eukaryotic cell division. In each
mitotic cell cycle, the centrosome must duplicate once in preparation
to form the spindle apparatus. The mitotic spindle is instrumental for
proper segregation of the duplicated chromosomes into two euploid
daughter cells, each receiving one of the centrosomes. Proper
centrosome function is vital to cell proliferation, and errors in
centrosome duplication, spindle formation, or spindle function can lead
to chromosome instability, chromosome non-disjunction, and aneuploidy.
Several lines of evidence from a wide variety of organisms suggest that
protein phosphorylation plays a major role in centrosome control during
the cell cycle. The MPM-2 monoclonal antibody recognizes mitosis-specific centrosomal phosphoepitopes in mammalian cells, Aspergillus nidulans and Schizosaccharomyces
pombe (1-3), and MPM-2 antibodies inhibit microtubule nucleation
in vitro (4). Several protein kinases have been implicated
in centrosome control by their mutant phenotype, including yeast Mps1p
(5, 6) and Drosophila aurora (7). In vertebrates,
CDK2/cyclinE is important for initiation of centrosome duplication
(8-10), and Nek2p is important for centrosome separation (11). Studies
involving phosphatases and phosphatase inhibitors indicate that
dephosphorylation is equally important for proper centrosome regulation
(12-15).
In the budding yeast S. cerevisiae, the spindle pole body
(SPB)1 is functionally
equivalent to the centrosome. The SPB is a multilayered cylinder
embedded in the nuclear envelope. Cytoplasmic microtubules emanate from
an outer plaque, and nuclear microtubules emanate from the inner
plaque. The 110-kDa spindle pole component Spc110p contains a large,
central coiled-coil domain, which is located in the region of the SPB
between the inner and central plaques (16). The C-terminal globular
domain of Spc110p is located at the central plaque of the SPB (17, 18),
and has been shown to interact with the SPB components calmodulin,
Spc29p and Spc42p (17, 19-22). The N-terminal globular domain of
Spc110p is located at the inner plaque, where Tub4p, Spc97p, and Spc98p
are found (18, 23). The N-terminal globular domain of Spc110p interacts both genetically and biochemically with these components of the Tub4p
complex (19, 23-25). Spc110p is also a phosphoprotein, and
phosphorylation of Spc110p at serine/threonine residues arises as cells
form the mitotic spindle and disappears as cells enter anaphase (26,
27).
The dual-specificity kinase Mps1p is essential for SPB duplication and
for mitotic checkpoint control in S. cerevisiae (6). Cells
harboring the temperature-sensitive mps1-1 allele fail to duplicate the SPB at the restrictive temperature yet proceed through a
doomed mitosis with a monopolar spindle (5). Mps1p is also involved in
the mitotic checkpoint (28). High levels of Mps1p cause cells to arrest
in metaphase, and these arrested cells contain hyperphosphorylated
Mad1p, which is also associated with activation of the mitotic
checkpoint (29). In vitro, GST-Mps1p phosphorylates Mad1p
(29) as well as Spc98p, a component of the Tub4p complex (30).
Despite the recent boon in centrosome component identification (for
example, see Ref. 31) and the implication that several kinases are
involved in centrosome function, direct evidence of specific
phosphorylation of a centrosome component by a specific kinase in
vivo has been lacking. In this study, we demonstrate that the
dual-specificity kinase Mps1p phosphorylates Spc110p in
vitro and that these sites are important for the mitosis-specific phosphorylation of Spc110p in vivo. These phosphorylations
occur within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and interacts with components of the
microtubule-organizing Tub4p complex. Mutating these phosphorylated residues to alanine (to prevent phosphorylation), but not to aspartate (to mimic phosphorylation), perturbs the function of Spc110p such that
it can no longer support growth in the presence of mutant forms of
Spc97p, a component of the Tub4p complex. This perturbation also
requires another alanine substitution at Ser36 in the
N-terminal globular domain, which is within an
(S/T)PX(R/K) consensus sequence for phosphorylation
by cdc2p/Cdc28p cyclin-dependent kinase (32, 33). Thus,
proper interaction of the Tub4p complex with the yeast centrosome may
require Spc110p phosphorylation at all four of these sites.
Media, Strains, and Genetic Manipulations--
SD complete,
SD-uracil (34), SD-uracil low adenine (17), YPD and YPD low adenine,
and LB (35) were described previously. SD-uracil+uracil is SD-uracil
supplemented with 25 µg/ml uracil. LB amp is LB medium supplemented
with 100 µg/ml ampicillin. LB amp kan is LB amp medium supplemented
with 6 µg/ml kanamycin. Plasmid transformations were carried out by
the LiOAc method essentially as described previously (36).
Strains are listed in Table I. The
spc110-4A allele was integrated into CRY1 by a two step gene
replacement (37) using plasmid pJK24 cut with SnaBI,
creating strain JKY1. The presence of the spc110-4A allele
was confirmed by sequencing. Synthetic lethal interactions between
spc110-4A and spc97-114 were tested by crossing
strain JKY1 (spc110-4A) with strain TNY64-5C
(spc97-114). Neither single mutant confers a
temperature-sensitive phenotype. The diploid was sporulated and the
tetrads dissected giving 25 tetratypes (1:3
temperature-sensitive:non-temperature-sensitive), 4 non-parental
ditypes (2:2), and 3 parental ditypes (0:4). Overall, 25.8% of the
spore clones from these dissections could not form colonies at
37 °C.
Immunoblot Analysis, Plasmids--
Plasmids are listed in Table
II. pCL5 expresses ArgU, an
arginine-tRNAAGA/AGG (AGA and AGG are rare codons in
Escherichia coli but common in yeast). pCL5 contains the
pA15 origin of replication to allow for co-expression with plasmids
containing the ColEI origin of replication. Plasmid pDV29
encoding GST-Spc110p-(1-183) (SPC110 GenBankTM accession
number Z11582) was constructed by cloning the
NcoI-EcoRI fragment from pDV17 (25) into the
SmaI and EcoRI sites of pGEX-2T (Amersham
Pharmacia Biotech, Piscataway, NJ). Plasmids pJK2, pJK4, and pJK7 were
made by site-directed mutagenesis of plasmid pDV29 using the USE kit
(Amersham Pharmacia Biotech) according to the manufacturer's
directions. All other pJK plasmids (except pJK24) were made by
site-directed mutagenesis of the parent plasmid using the QuikChange
kit (Stratagene, La Jolla, CA) according to the manufacturer's
directions. Plasmid pJK21 carrying URA3, CEN, and
an spc110 allele was converted to integrating plasmid pJK24
by replacing the AlwNI fragment containing part of
bla, CEN, and part of URA3 with
the AlwNI fragment from pRS306 containing part of
bla and part of URA3 but no CEN.
Plasmids pDF47 and pDF48, which express 12xHIS-Spc110p fusion proteins,
were constructed in several steps. First, a 6xHis tag was inserted at
the initiator MET of SPC110 in plasmid pHS31 by
site-directed mutagenesis as described in a previous study (26),
creating pDF18. Plasmid pDF29 encodes the functional
SPC110-201 allele containing an in-frame deletion of the
coding sequences for residues 267-543 within the central coiled-coil
(26). Plasmid pDF30 (6xHIS-SPC110-201) was constructed by
swapping a 1.6-kb HindIII fragment from pDF29 into pDF18.
pDF47 expresses 12xHIS-Spc110-201p(756 Production of Spc110p Fusion Proteins--
Cultures of E. coli strain GM1 were transformed to ampicillin resistance with
plasmids pDF47 or pDF48 expressing recombinant 12xHIS-Spc110-201p(756 Mps1p in Vitro Kinase Assay--
All reactions were carried out
as described previously (6) except as follows. Kinase assays used to
generate phosphorylated GST-Spc110p-(1-183) for analysis
of phosphorylation sites by mass spectrometry used 200 µl of
GST-Mps1p bound to GSH-Sepharose in a 50% slurry, which was washed
once in kinase assay buffer without ATP (KAB-ATP: 50 mM
Tris, pH 7.5, 10 mM MgCl2, 2 mM
DTT) and resuspended in 50 µl of KAB-ATP and 10 µl of
GST-Spc110p-(1-183) at a concentration of 1 mg/ml. 40 µl of 5× KAB
(final concentration, 50 mM Tris, pH 7.5, 10 mM
MgCl2, 2 mM DTT, 2 mM ATP
containing 200 µCi of [ Proteolytic Digestion and Mass Spectrometry--
Both kinase and
substrate were expressed as GST fusions for these experiments, and both
were bound to GSH-Sepharose resin during the reaction. Prior to
proteolytic digestion the resin was washed twice and resuspended in 100 µl of 50 mM Tris, pH 7.5, 1 mM
CaCl2 for digestion with trypsin, or in 100 µl of 100 mM Tris, pH 9.2, for digestion with endoproteinase Lys C
(endoLys-C). Proteolytic digestion was performed at 30 °C overnight
with shaking to keep the resin in suspension. The resin was removed by
centrifugation, leaving the peptides in the supernatant, then washed
once with 50 µl of H2O. The wash and supernatant were
combined, reduced in a vacuum-concentrating microcentrifuge
(Heto-Holten, Allerød, Denmark) to near dryness, and resuspended in 50 µl of 0.1-1.0% trifluoroacetic acid. Electrospray ionization,
liquid chromatography mass spectrometry (ESI-LC/MS), and tandem mass
spectrometry (ESI-LC/MS/MS) were carried out using either a hand-packed
500-µm i.d. HPLC column containing C18 reverse phase resin (Columbus)
interfaced to an API III+ triple-quadrupole mass spectrometer
(PE-Biosystems, Foster City, CA), or a 320-µm i.d. HPLC column
(Micro-Tech, Sunnyvale, CA) packed with C18 reverse phase resin
interfaced to an LCQ ion trap mass spectrometer (ThermoQuest, San Jose,
CA). Matrix-assisted laser desorption ionization, time of flight
(MALDI-TOF) mass spectrometry was carried out on 0.5 µl of analyte
mixed with 0.5 µl of Mps1p Activity Is Necessary for Mitosis-specific Phosphorylation of
Spc110p in Vivo--
Previously we have shown that the 110-kDa SPB
component Spc110p undergoes serine/threonine phosphorylation in cells
containing pre-anaphase mitotic spindles, which results in a
slower-migrating isoform (p120) during SDS-PAGE (26). We analyzed the
Spc110p mobility shift in cells carrying the mps1-1 mutation
at the restrictive temperature to determine whether the essential
dual-specificity kinase Mps1p played a role in the mitosis-specific
Spc110p phosphorylation. Mps1-1p is a severely crippled conditional
mutant kinase, both in vivo and in vitro (38).
Cells were first synchronized in G1 by the addition of the
mating pheromone
MPS1 has at least two previously identified execution points
during the cell cycle, one that is essential for SPB duplication (5),
and another that is involved in the mitotic spindle checkpoint (28).
Because the mitosis-specific phosphorylation of Spc110p does not occur
until after SPB duplication is complete (26), it is possible that the
dependence for Spc110p phosphorylation on Mps1p function merely
reflects a prerequisite for SPB duplication. To test this hypothesis,
we performed similar analyses for Spc110p phosphorylation at the
restrictive temperature in two additional mutant backgrounds that block
SPB duplication, mps2-1 and cdc31-2. Both
mutations cause cells to arrest with large buds and a G2 DNA content at the restrictive temperature. Cells carrying the mps2-1 mutation arrest at the restrictive temperature with a
malformed duplicated SPB that fails to insert into the nuclear envelope and lacks both the inner plaque and nuclear microtubules (5). Cells
carrying the cdc31-2 mutation fail completely in SPB
duplication (39). The slower migrating mitosis-specific isoform of
Spc110p is present at the restrictive temperature in each mutant
background despite failures in SPB duplication (Fig.
2). Furthermore, ordering of execution
points during the cell cycle place CDC31 function first,
followed by MPS1 function and then by MPS2
function (5). Thus failure of the mitosis-specific phosphorylation of
Spc110p in mps1-1 cells is not due simply to a failure in
SPB duplication.
We also tested if MPS1 expression could promote the
production of the mitosis-specific Spc110p isoform in cells blocked in G1 by the addition of Mps1p Phosphorylates Spc110p in Vitro at Sites within the
N-terminal Globular Domain of Spc110p--
We expressed different
forms of recombinant Spc110p in E. coli, and the lysates
were added to an in vitro GST-Mps1p kinase assay (6).
GST-Mps1p (purified from yeast) phosphorylates
12xHIS-Spc110-201p(756
Trypsin digestion followed by two-dimensional TLE/TLC phosphopeptide
mapping was used to determine the complexity of Spc110p phosphorylation
by GST-Mps1p. The two-dimensional phosphopeptide maps of
12xHIS-Spc110-201p(756 Ser60, Thr64, and Thr68 in
Spc110p Are Phosphorylated by GST-Mps1p in Vitro--
We used an
E. coli-expressed GST-Spc110p -(1-183) fusion
protein containing the first 183 residues of Spc110p as a substrate for
GST-Mps1p to determine the in vitro sites of phosphorylation by GST-Mps1p within the N-terminal globular domain of Spc110p. Stoichiometry of phosphorylation in three experiments ranged from 0.9 to 1.7 mol of phosphate per mol of GST-Spc110p-(1-183), and this
phosphorylation produced a mobility shift of GST-Spc110p-(1-183) on
SDS-PAGE (Fig. 5A).
The phosphorylated GST-Spc110p-(1-183) was digested with trypsin or
endoLys-C, and the peptides were analyzed by MALDI-TOF and ESI-LC/MS
mass spectrometry. This analysis accounted for every serine, threonine,
and tyrosine residue within the first 183 residues of Spc110p, and
identified four tryptic peptides and two endoLys-C peptides that
appeared to be phosphopeptides based on mass increases of 80 Da (or
multiples thereof) over the expected masses for these peptides. All
candidate phosphopeptides encompassed the residues 60SIDDTIDSTR69 within the N-terminal
globular domain of Spc110p. Two representative fragmentation spectra of
these phosphopeptides are described below.
The tryptic fragment 60SIDDTIDSTR69 (Tp60-69)
was found to contain a single phosphate. Sequencing this peptide by
collision-induced dissociation during ESI-LC/MS/MS confirmed this
assignment and mapped the site of phosphorylation to residue
Thr64 (Fig. 5B). This peptide, containing
phosphorylation at Thr64, was also identified from the
lowest of the three spots in the two-dimensional phosphopeptide maps
shown in Fig. 3 (data not shown). In the endoLys-C digest, mono- and
di-phosphorylated forms of the peptide
56RQRRSIDDTIDSTRLFSEASQFDDSFPEIK85 (K56-85)
were observed. The fragmentation spectrum of the di-phosphorylated form
of K56-85 by ESI-LC/MS/MS showed heterogeneity in phosphorylation at
any two of the three residues Ser60, Thr64, and
Thr68 (Fig. 5, C and D). Several
fragment ions indicated both phosphorylated (addition of
HPO4 or neutral loss of H3PO4) as
well as unmodified Ser60 (denoted by the
asterisk in Fig. 5C and detailed in Fig.
5D). Fragment ions containing all three residues
Ser60, Thr64, and Thr68 were found
in the mono- or di-phosphorylated forms, but never at their expected,
unmodified masses. Thus, a mixture of di-phosphorylated forms of this
peptide (phosphorylated at any two of these three sites) co-eluted from
the reverse phase resin and were fragmented simultaneously during the
LC/MS/MS experiment. A tri-phosphorylated form of K56-85 was not found.
Sites of in vitro Spc110p phosphorylation by Mps1p
were confirmed by repeating the kinase assays using
GST-Spc110p-(1-183) fusions containing combinations of
alanine substitutions at residues Ser60, Thr64,
and Thr68 (Fig. 6). The level
of phosphate incorporation for each single mutant was decreased
relative to the wild-type level (80%, 40%, and 50% that of the
wild-type level for the single mutants S60A (not shown in Fig. 6),
T64A, and T68A, respectively). The level of phosphate incorporation
into the T64A,T68A double mutant was 20% that of the wild-type level,
and the level for the triple mutant S60A,T64A,T68A was reduced to
near background levels (Fig. 6).
The kinase assays using mutant GST-Spc110p-(1-183) fusions as
substrates confirmed phosphorylation at Thr68. The MS/MS
spectrum for K56-85 does not clearly distinguish between phosphorylation at Ser67 and Thr68 (Fig.
5C and data not shown). However, phosphorylation of
GST-Spc110p-(1-183) containing the T68A mutation was reduced relative
to the wild-type protein as stated above, whereas the same fusion
protein containing the S67A mutation was indistinguishable from the
wild-type protein in this assay (data not shown). In addition, the
mobility shift of GST-Spc110p-(1-183) observed during the in
vitro kinase assay was dependent upon the presence of
Thr68 (Fig. 6).
S60A, T64A, or T68A Mutations, in Conjunction with an S36A
Mutation, Are Synthetically Lethal with Mutations in SPC97--
The
spc110-221 allele, which contains mutations in the
N-terminal globular domain of Spc110p (19), is synthetically lethal with alleles of SPC97 and SPC98 (25). We find
that full-length Spc110p containing any of the S60A, T64A, or T68A
mutations, in conjunction with an alanine substitution at
Ser36, fails to complement the synthetic lethality between
spc110-221 and spc97-62 or spc97-114
(Table III, lines 5-9). We
have also found that Ser36 is phosphorylated in Spc110p
when expressed in insect
cells.2 The S36A substitution
alone was still able to complement spc110-221 spc97
synthetic lethality, as was the triple Mps1p site substitution (Table
III, lines 2-4). Full-length Spc110p containing aspartate substitution at Ser36, Ser60,
Thr64, and Thr68 was also able to complement
the synthetic lethality (Table III, lines 10-11),
suggesting that the effects due to the alanine substitutions were not
due to an overall perturbation of protein structure. In contrast, the
synthetic lethality between spc110-221 and an allele of
Spc98p (spc98-63) was still suppressed by the quadruple alanine substitution Spc110p (Table III, line 9).
These results were confirmed by integrating the spc110-4A
allele containing the S36A, S60A, T64A, and T68A mutations and testing directly for synthetic lethal interactions with spc97-114
("Experimental Procedures"). The double mutant
(spc110-4A and spc97-114) was not viable at
32 °C, whereas neither allele alone (spc110-4A or spc97-114) conferred a temperature-sensitive phenotype.
These mutant proteins were assayed by Western blot analysis when
expressed in strain HSY2-12C to assess their effect on the Spc110p
mitosis-specific SDS-PAGE mobility shift. An asynchronous wild-type
culture exhibits both Spc110p isoforms (Fig. 2, lanes 1 and 6; Fig. 3, lane 1; Fig.
7 lane 1), whereas the
mitosis-specific mobility shift of Spc110p containing alanine
substitutions at the three Mps1p phosphorylation sites
(S60A,T64A,T68A) was abolished (Fig. 7, lane
4). The complete loss of the slower-migrating mitosis-specific isoform was dependent upon simultaneous mutation of all three Mps1p
phosphorylation sites (Fig. 7, lanes 4 and 5 and
data not shown) and was independent of alanine substitution at
Ser36. The mobility shift of the quadruple alanine
substitution mutant S36A,S60A,T64A,T68A Spc110p was similarly
abolished (Fig. 7, lane 3, and Fig. 3, lane 2),
and alanine substitution at only Ser36 did not markedly
affect the slower-migrating mitosis-specific isoform (Fig. 7,
lane 6). Simultaneous aspartate substitution at these same
four residues resulted in the complete shift of Spc110p into a
slower-migrating form (Fig. 7, lane 2), but otherwise did
not affect Spc110p function.
We have shown previously that the mitosis-specific
serine/threonine phosphorylation of Spc110p occurs after SPB
duplication is completed as the mitotic spindle first forms, and
persists up to the metaphase/anaphase transition (26). We show here
that the dual-specificity kinase Mps1p phosphorylates Spc110p and that this phosphorylation is necessary for the production of the
mitosis-specific Spc110p isoform in vivo. Mitosis-specific
Spc110p phosphorylation can occur in the absence of proper SPB
duplication but cannot occur in the absence of Mps1p activity, and
Mps1p production during G1 arrest can drive formation of
the mitosis-specific Spc110p isoform. Spc110p is phosphorylated at
residues Ser60, Thr64, and Thr68 by
GST-Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific phosphorylation in vivo.
These sites of phosphorylation are in the N-terminal globular domain of
Spc110p, which resides at the inner plaque of the SPB and interacts with members of the microtubule-nucleating Tub4p complex (19, 23-25).
In vivo, alanine substitution at the Mps1p phosphorylation
sites in Spc110p is synthetically lethal with alleles of
SPC97, which encodes a component of the Tub4p complex. This
synthetic lethality also requires an additional alanine substitution at residue Ser36. Ser36 falls within an
(S/T)PX(R/K) consensus sequence for phosphorylation by
cdc2p/Cdc28p cyclin-dependent kinase (32, 33), and
Ser36 is phosphorylated when Spc110p is purified from
insect cells.2 We have shown previously that
Ser36 does not contribute to the mitosis-specific Spc110p
isoform (26), and those findings are reiterated in this study (Fig. 7,
lane 6). However, the S36A mutation is required for
synthetic lethality with alleles of SPC97, and, in the
presence of the S36A mutation, alanine substitution at any one of the
three Mps1p sites is all that is required to produce the synthetic
lethal phenotype.
Simultaneous aspartate substitution at Ser36 and the Mps1p
sites does not interfere with Spc110p function, including the ability to complement synthetic lethality between alleles of spc110
and spc97. Whereas alanine substitution at serine and
threonine residues is thought to prohibit side-chain phosphorylation
without perturbing overall protein structure, aspartate substitutions
at these residues are thought to mimic phosphorylation owing to the
bulky, negatively charged aspartate residue side chain. Thus the
phenotype associated with the alanine substitution Spc110p is not due
simply to an overall perturbation of protein structure, but more likely
to a specific defect due to the loss of phosphorylation at these residues.
The mitosis-specific phosphorylation of Spc110p occurs at a time when
spindles first form (26), and alleles containing alanine substitutions
at Ser36 and the Mps1p sites perturb the interaction
between Spc110p and Spc97p. It is tantalizing to suggest that Spc110p
phosphorylation is important for proper interaction with the Tub4p
complex. However, it is important to note that the spc97-114
allele exhibits a number of genetic interactions both with mutations in
SPC110 and with genes encoding components of the Tub4p
complex (25). Thus the synthetic lethality between spc110-4A
and spc97-114 reflects a defect in Spc110p function but does
not specify which function of Spc110p is compromised. In contrast, the
spc110-4A allele was still able to complement synthetic
lethality between spc110-221 and spc98-63 (Table
III, line 9). The fact that the spc98-63 allele tested here was unperturbed by the spc110-4A allele is not
surprising, because spc98-63 has defects that are specific
to the spc110-221 and spc110-222 alleles and
exhibits no genetic interactions with other mutations in
SPC110 or with mutations in the Tub4p complex components
(25).3
Mps1p has a demonstrated role in both SPB duplication and the mitotic
spindle checkpoint (6, 28, 29). A role for Mps1p during mitotic spindle
formation has been suggested by genetic interactions with the
CIN8-encoded kinesin-like protein (40), and the spindle
component encoded by DAM1 (41). Our results here confirm a
third role for Mps1p activity during spindle formation and suggest that
it is important for Spc110p function. Consistent with this third Mps1p
role demonstrated herein, Mps1p kinase isolated from synchronized
cycling cells exhibits a peak of activity at the same time Spc110p
phosphorylation occurs as the mitotic spindle forms.4
Mps1p phosphorylation of the Spc110p N-terminal globular domain may
modulate the interaction between the microtubule-nucleating Tub4p
complex and the SPB. The regions encompassing sites of Spc110p phosphorylation may form a docking site for the Tub4p complex or
provide access to the docking site when phosphorylated. Another possible role for this phosphorylation is modulation of microtubule dynamics during mitotic spindle formation, which may also occur through
additional phosphorylation of Tub4p complex components by Mps1p.
Indeed, the Tub4p complex component Spc98p is also a phosphoprotein
that exhibits mitosis-specific phosphorylation similar to that seen for
Spc110p, and GST-Mps1p can phosphorylate Spc98p in vitro
(30). Phosphorylation of Spc98p may complement the phosphorylation of
Spc110p in the binding of the Tub4p complex such that mutations in the
phosphorylation sites of both of these binding partners would have
catastrophic consequences. Overall, Mps1p phosphorylation of proteins
involved in microtubule nucleation during mitosis may be a mechanism by
which Mps1p contributes to the assembly and stability of the SPB and
the mitotic spindle that it forms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains used in this
study
-Factor Arrest, and Cytological
Techniques--
Immunoblot analysis,
-factor arrests, cell
morphology characterization, and flow cytometry were performed as
described (26).
) (missing residues 267-543
within the central coiled-coil and truncated at residue 756 of Spc110p)
and was constructed by cloning the SphI-SspI
fragment from pDF30 into the SphI and SmaI sites
of the 6xHis bacterial expression vector pQE32. pDF48 expresses
12xHIS-Spc110 P-(1-225) (truncated at residue 225 of Spc110p) and was
constructed by cloning the SphI-NsiI fragment
from pDF30 into the SphI and PstI sites of
pQE32.
Plasmids used in this study
) or 12xHIS-Spc110p-(1-225), respectively. Cultures of isolated transformants were diluted 1:100 into 10 ml of LB
amp, grown to a density of 20 Klett units and then induced with 1-2
mM isopropyl-1-thio-
-D-galactopyranoside
for 5 h at 30 °C. Cells were harvested, washed once in lysis
buffer (1× phosphate-buffered saline, 10 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and lysed using a French Pressure cell (3/8-inch piston diameter; Aminco) at 11,000 p.s.i. to near 100% lysis. Wild-type and mutant forms of recombinant GST-Spc110p-(1-183) were co-expressed with plasmid pCL5 in strain GM1. Overnight cultures were diluted 1:200 into
200 ml of LB amp kan and grown at 37 °C to a density of 60-70 Klett
units. Isopropyl-1-thio-
-D-galactopyranoside was added to a final concentration of 40 µg/ml, and cultures were incubated for
an additional 1.5 h. Cells were then pelleted at 8500 × g for 10 min, and pellets were stored at
80 °C. Thawed
pellets were suspended in 3 ml of cold lysis buffer (1×
phosphate-buffered saline, 10 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and lysed in a French
Pressure cell as described above. Triton X-100 was added to a final
concentration of 1%, and the lysate was incubated on ice for 15 min.
Cleared lysates were mixed for 1 h with 100 µl of glutathione
Sepharose 4B resin (Amersham Pharmacia Biotech). The supernatant was
then discarded, and the beads were washed three times with 1 ml of
lysis buffer. GST-Spc110p-(1-183) was eluted in 200 µl of elution
buffer (10 mM glutathione, 50 mM Tris, pH 8.0)
by mixing for 10 min. The buffer was exchanged to storage buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.5 mM DTT, 5% v/v glycerol) by gel
filtration on G-25 Sephadex. Samples were frozen in liquid
N2 and stored at
80 °C.
-32P]ATP) was added, and the
mixture was incubated at 30 °C with shaking for 5 h. 20 µl
was subjected to 12% SDS-PAGE. The gel was stained with Coomassie
Brilliant Blue, dried, and quantified by PhosphorImager analysis to
assess stoichiometry of phosphate incorporation. Kinase assays used to
compare incorporation of phosphate into wild-type and phosphorylation
site mutants of GST-Spc110p-(1-183) used 10 µl of
GST-Mps1p conjugated to GSH-Sepharose beads and ~0.2 µg of
GST-Spc110p-(1-183) (wild-type or mutant). The entire reaction was
separated by SDS-PAGE, and the gel was stained with Coomassie Brilliant
Blue prior to PhosphorImager analysis.
-cyano-4-hydroxycinnamic acid matrix (Agilent
Technologies, Hewlett-Packard), using a Voyager DE-STR mass
spectrometer (PerSeptive Biosystems, Foster City, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor and then released into prewarmed 37 °C
medium. At 37 °C, mps1-1 cells entered the cell cycle at
the same time as the mps1-1 pMPS1 control cells (as
evidenced by bud emergence at 30 min after release for both cultures),
but unlike the wild-type control, the slower migrating mitosis-specific
isoform of Spc110p never accumulated in mps1-1 cells (Fig.
1). Thus, MPS1 is required for
the mitosis-specific phosphorylation of Spc110p in vivo.
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Fig. 1.
Mitosis-specific Spc110p phosphorylation is
dependent upon Mps1p function. Strain Wx241-2b
(mps1-1), alone or transformed with plasmid pMPS1, was grown
at room temperature in SD-uracil and SD-uracil+uracil medium,
respectively (doubling time = 3.5 h). Mid-logarithmic
cultures were exchanged into YPD medium. Cultures were then treated
with 6 µM -factor at room temperature for 2.5 h,
after which cultures were shifted to 37 °C. After an additional
1.75 h of
-factor treatment at 37 °C (>95% shmoo
morphology), cultures were released from the arrest into
prewarmed YPD at 37 °C, and aliquots were taken for total cell
protein (trichloroacetic acid precipitation), bud morphology, and DNA
content at the indicated time points as described (26). A,
protein content and bud morphology. Western blot analysis using
affinity-purified anti-Spc110p antibodies was performed as described
previously (26). 120- (p120) and 112-kDa (p112)
Spc110p isoforms are indicated. Buds appeared 30 min after release from
-factor for both cultures. Lanes 1-7, mps1-1
cells; lanes 8-14, mps1-1 cells harboring
plasmid pMPS1. B, DNA content. The time points taken just
prior to release from G1 arrest (37 G1 arrest
(0)) were taken at the same time as the 0-min time points shown in
A. Additional aliquots were taken during asynchronous growth
at room temperature (RT asynch) and G1 arrest at
room temperature (RT G1 arrest) to establish the
distribution of cells containing haploid DNA content (1N)
and diploid DNA content (2N). Peak height reflects cell
number. By the last time point (180 min after release) cells carrying
the pMPS1 plasmid had returned to G1 as assessed by DNA
content, whereas the mps1-1 cells exhibited a wide range of
DNA content, including below 1N and above 4N as
observed previously (5).
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Fig. 2.
Mitosis-specific Spc110p phosphorylation
persists in the absence of SPB duplication. Western blot analysis
using affinity-purified anti-Spc110p antibodies was performed as
described previously (26). Strains Wx178-3A and Wx209-8A carrying the
mutations mps2-1 or cdc31-2, respectively, were
each grown at room temperature to mid-logarithmic phase (doubling
times = 2.67 and 3 h, respectively). Each culture was then
shifted to the restrictive temperature of 37 °C for 4.5 h,
after which total cell protein extracts were obtained from 1-ml
aliquots by trichloroacetic acid precipitation as described (26).
Separate cultures were treated with 6 µM -factor at
room temperature, and total cell protein extracts were obtained from
1-ml aliquots after 1.5 doublings (>96% shmoo morphology). The
cultures were then washed with prewarmed YPD and released from the
-factor arrest in YPD at 37 °C, and total cell extracts were
prepared from 1 ml of culture after the indicated time at 37 °C.
Lanes 1-5, mps2-1 cells grown at room
temperature for 4.5 h (lane 1), incubated at 37 °C
for 4.5 h (lane 2), arrested with
-factor at room
temperature (lane 3), and released from
-factor arrest at
37 °C for 2 h or 3 h (lanes 4 or 5).
Lanes 6-11: cdc31-2 cells grown at room
temperature for 4.5 h (lane 6), incubated at 37 °C
for 4.5 h (lane 7), arrested with
-factor at room
temperature (lane 8), and released from
-factor arrest at
37 °C for 2, 3, or 4.5 h (lanes 9-11). DNA content
was consistent with previously published findings for mps2-1
and cdc31-2 at the restrictive temperature (5, 39). 120- (p120) and 112-kDa (p112) Spc110p isoforms are
indicated.
-factor, a state where the
mitosis-specific isoform is normally not present (26). Cells expressing
MPS1 from a galactose-inducible promoter while held at this
G1 arrest clearly produce the mitosis-specific Spc110p
isoform (Fig. 3, lane 9),
whereas similarly arrested cells harboring control plasmids do not
(Fig. 3, lane 7). A non-phosphorylatable form of Spc110p (4A, described below) is not shifted in this experiment
(Fig. 3, lane 10), demonstrating that the Spc110p mobility
shift promoted by the expression of MPS1 during
G1 arrest does not result from phosphorylation at
inappropriate sites. Because other mitosis-specific functions would be
turned off during this G1 arrest, it is likely that the
production of the slower-migrating Spc110p isoform, which normally
results from mitosis-specific phosphorylation, here results directly
from the inappropriate expression of Mps1p.
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Fig. 3.
Mps1p expression during G1 arrest
results in the production of the mitosis-specific Spc110p isoform.
Western blot analysis using affinity-purified anti-Spc110p antibodies
and preparation of total cell protein extracts was performed as in Fig.
1. CRY1 (wt) and JKY1 (4A) harboring either
plasmid pEGKT (pGAL1-GST) or plasmid pEGKTMps1 2
(pGAL1-GST-MPS1) were grown under the following conditions.
Galactose induction during the G1 arrest was performed as
described previously (29) with the exception that cells were held at
the arrest during the induction by the further addition of 10 µM
-factor after 2 h. Lanes 1 and
2, asynchronous cultures. Lanes 3-6, cells
arrested during the G1 stage of the cell cycle by the
addition of 10 µM
-factor. Lanes 7-10,
galactose induction during the G1 arrest. 120- (p120) and 112-kDa (p112) Spc110p isoforms are
indicated. The high mobility signal present in lanes 9 and
10 results from the galactose-induced expression of
GST-Mps1p and is visualized due to cross-reactivity between the
affinity-purified anti-Spc110p antibodies (raised against a GST fusion
to the coiled-coil region of Spc110p (26)) and the GST-Mps1p fusion
protein. The apparent molecular mass of GST-Mps1p in lanes
9 and 10 is 150 kDa, which is in agreement with
previous findings (6, 38) despite a predicted molecular mass of 112 kDa. Furthermore, the 150-kDa band in lanes 9 and
10 is the only protein reacting with anti-GST antibodies in
a Western blot analysis (data not shown).
), which is missing residues 267-543 within
the central coiled-coil region and the last 188 residues of the
C-terminal globular domain (Fig.
4A, lane 2).
12xHIS-Spc110p-(1-225), which consists of the first 225 amino acids
and encompasses the N-terminal globular domain of Spc110p, was also
phosphorylated in vitro by GST-Mps1p (Fig. 4A,
lane 3). A fusion containing a portion of the central coiled-coil (residues 265-755) was not phosphorylated by GST-Mps1p (Fig. 4A, lane 9). Phosphorylation of the various
Spc110p substrates depended on the addition of GST-Mps1p and was
distinct from any background phosphorylation from the E. coli extract (Fig. 4A, lane 1), or from any
breakdown products resulting from Mps1p autophosphorylation (Fig.
4A, lane 4).
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Fig. 4.
In vitro phosphorylation of
Spc110p by Mps1p. A, GST-Mps1p in vitro
kinase assays. Crude bacterial extract from cultures expressing
recombinant 12xHIS-Spc110-201p(756 ) (pDF47),
12xHIS-Spc110p-(1-225) (pDF48), or no fusion protein (pQE32) was used
as substrates for phosphorylation by GST-Mps1p kinase (purified from
yeast) in the presence of radiolabeled [
-32P]ATP as
described under "Experimental Procedures." Purified
6xHIS-Spc110p-(265-755), containing residues 265-755 within the
central coiled-coil (26), and myelin basic protein (MBP)
were also used as substrates. Kinase assays were boiled for 4 min in
SDS-PAGE buffer prior to separation on 12% SDS-polyacrylamide gels
(42). The dried gels were exposed to autoradiography film or a
PhosphorImager screen. Lane 1, 12xHIS-Spc110-201p(756
)
expressed from plasmid pDF47 with no GST-Mps1p added to the reaction.
Lane 2, 12xHIS-Spc110-201p(756
) expressed from plasmid
pDF47 with GST-Mps1p added to the reaction. Lane 3,
12xHIS-Spc110p-(1-225) expressed from plasmid pDF48 with GST-Mps1p
added to the reaction. Lane 4, expression plasmid pQE32
containing no fusion protein with GST-Mps1p added to the reaction.
Lanes 5 and 6, Western blot analysis using
anti-MRGS antibodies (Qiagen, Valencia, CA) as described previously
(26) to determine the mobility of 12xHIS-Spc110-201p(756
) expressed
from plasmid pDF47 (lane 5) and 12xHIS-Spc110-(1-225)
expressed from plasmid pDF48 (lane 6). Lane 7, no
substrates added to the reaction. Lane 8, MBP added to the
reaction. Lane 9, 6xHIS-Spc110p-(265-755) added to the
reaction. Lanes 10-12, Coomassie Brilliant Blue-stained gel
shown for lanes 7-9. The relative mobilities of the kinase
and substrates are indicated on the sides of the gels.
Asterisks in lanes 2 and 3 mark
positions of radiolabeled bands excised for phosphopeptide mapping.
B and C, two-dimensional thin layer
electrophoresis (1st), thin layer chromatography
(2nd; two-dimensional TLE/TLC) phosphopeptide mapping.
Open arrowheads denote origins. Proteins in gels similar to
A were electrophoretically transferred to nitrocellulose.
Bands corresponding to full-length 12xHIS-Spc110-201p(756
) and
12xHIS-Spc110p-(1-225) (asterisks in panel A)
were excised from the nitrocellulose support after comparison to an
autoradiogram of the same membrane. Phosphopeptides were then subjected
to two-dimensional TLE/TLC after digestion of the proteins off of the
nitrocellulose membrane using the protease trypsin as described in
(43). Shown are the tryptic phosphopeptide maps of
12xHIS-Spc110-201p(756
) (B) and 12xHIS-Spc110-(1-225)
(C) after phosphorylation by GST-Mps1p. D,
graphical representation of the recombinant proteins with respect to
full-length Spc110p (containing 944 residues). The shaded
area represents the central coiled-coil domain (residues 155-798
as predicted by PAIRCOIL). Fusions containing the C-terminal domain
were insoluble and could not be tested. However, serine and threonine
residues within the C-terminal 116 amino acids have been shown
previously not to be involved in the mitosis-specific phosphorylation
of Spc110p (26), leaving eight serines and/or threonines between
residues 756 and 828 that have not been tested.
) and 12xHIS-Spc110p-(1-225) were nearly
identical, exhibiting three major 32P-labeled
phosphopeptides and varying only in weak background phosphorylation
(Fig. 4, B and C). Thus, the major
phosphorylation of Spc110p by GST-Mps1p resides in the N-terminal
globular domain of Spc110p. This domain of Spc110p resides at the inner
plaque of the SPB and associates with the microtubule-organizing Tub4p complex containing Spc97p, Spc98p, and Tub4p (23-25).
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Fig. 5.
Mps1p phosphorylates Spc110p at residues
Ser60, Thr64, and Thr68 in
vitro. A, GST-Mps1p in vitro
kinase assay. GST-Spc110p-(1-183) was purified from E. coli
and subjected to phosphorylation by GST-Mps1p in vitro as
described under "Experimental Procedures." Lane 1,
GST-Spc110p-(1-183) (0.2 µg) with no GST-Mps1p kinase added to the
reaction. Lane 2, same as lane 1 with the
addition of GST-Mps1p as described under "Experimental Procedures."
The GST-Spc110p-(1-183) mobility shift is accompanied by 1.4 mol/mol
of phosphate incorporation. Tryptic or endoLys-C peptides were obtained
from phosphorylated GST-Spc110p-(1-183) after incubation with protease
as described under "Experimental Procedures," and each peptide
mixture was analyzed by ESI-LC/MS in positive ion mode. Scans across
the entire LC range were surveyed for each peptide mass including
mass-shifts of 80 Da to account for phosphopeptides. B and
C, MS/MS peptide sequencing of two phosphopeptides. Peptide
bond cleavages generating b ions (containing the N terminus) and y ions
(containing the C terminus) are illustrated. A summary of the observed
fragmentation ions is shown for the two spectra, including
m/z shifts consistent with phosphorylation (P and
PP) and neutral loss of H3PO4
( and
). Singly, doubly, or triply
charged b ions (b, b2+, and b3+) are displayed
above the sequence (
), and singly or doubly charged y ions (y and
y2+) are displayed below (
). B, MS/MS
spectrum of tryptic fragment 60SIDDTIDSTR69 + 80 Da (one phosphate; (M+2H)2+ = 602.0 Da/e). This spectrum
represents the summation of nine scans that were acquired as the
phosphopeptide eluted from the HPLC column. Singly charged y1, y3, y4,
and y5 ions were found at their expected masses based on the amino acid
sequence (175.4, 362.8, 478.6, and 590.2 Da/e, respectively). y6, y7,
and y8 ions were also found, but only with m/z shifts
consistent with phosphorylation (Py7 = 888.2 Da/e;
Py8 = 1002.8 Da/e) or neutral loss of
H3PO4 (
y6 = 675.4 Da/e;
y7 = 788.8 Da/e;
y8 = 904.6 Da/e). Ions consistent with
phosphorylation appear in the y-ion series beginning with ion y6,
indicating Thr64 as the phosphorylated residue in this
peptide. C, MS/MS spectrum of endoLys-C peptide
56RQRRSIDDTIDSTRLFSEASQFDDSFPEIK85 + 160 Da
(two phosphates; (M+4H)4+ = 931.0 Da/e). The m/z
of the two H3PO4 neutral loss products of the
parent ion are labeled (906.6 and 882.1 Da/e). For doubly charged
fragment ions, symbols indicate ions shifted in m/z from
that predicted by the amino acid sequence by +40, +80,
9, or
18 Da,
representing the addition of one or two phosphates (P or
PP) or the neutral loss of one or two
H3PO4 groups (
or
). The same changes in the triply charged fragment
ions create m/z shifts of +26.7, +53.3, or
12 Da,
representing the addition of one or two phosphates (P or
PP) or the neutral loss of two H3PO4
groups (
). Doubly and triply charged b ion cleavages
past Thr68 are found in either the mono- or
di-phosphorylated forms only. Asterisks indicate both
phosphorylated and unmodified ions were observed, as detailed in
D. Ion signals stronger than 2.5× background noise are
included in the summary above the spectrum. Weaker fragment ions
indicating phosphorylation at Thr68 and not
Ser67 were found but not labeled. D, expansion
of the spectrum shown in C from 400 to 800 Da/e detailing
ions consistent with a mixture of phosphorylated and unphosphorylated
Ser60. Symbols indicate doubly charged ions shifted in
m/z from that predicted by the amino acid sequence by +40,
9, or
18 Da, representing the addition of phosphate (P)
or the neutral loss of one (
) or two (
)
H3PO4 groups. The expected mass of the
unmodified doubly charged ion b72+ was found
(456.5 Da/e) along with ions consistent with phosphorylation
(Pb72+, 496.9 Da/e) and neutral loss of
H3PO4 (
b72+, 448.0 Da/e). The same pattern was found for b82+ ions
(505.3, 514.2, and 554.2 Da/e for ions
b82+,
b82+, and Pb82+,
respectively). Also shown are b62+ (398.9 Da/e)
and Pb62+ (439.2 Da/e). Cleavages creating the
b92+, b102+, and
b112+ ions include both Ser60 and
Thr64, and only the mono-phosphorylated forms are found for
these ions (Pb92+ = 604.6 Da/e,
Pb102+ = 661.3 Da/e,
Pb112+ = 718.8 Da/e,
b112+ = 670.1 Da/e). Shown is the
double-neutral loss ion
b132+ at 755.0 Da/e. Nearly every peak is accounted for despite ion signals being
close to 2.5× background noise in this region.
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Fig. 6.
Mutation of Spc110p phosphorylation sites
abolishes phosphorylation by Mps1p in vitro.
GST-Spc110p-(1-183) containing alanine substitutions at the indicated
residues were constructed and purified as described under
"Experimental Procedures." Approximately 0.2 µg were used as
substrates for in vitro GST-Mps1p kinase assays as described
under "Experimental Procedures," after which reactions were boiled
for 4 min in SDS-PAGE buffer prior to separation through a 10%
SDS-polyacrylamide gel (42). The gel was stained with Coomassie
Brilliant Blue (A), dried, and exposed to a PhosphorImager
screen (B). Arrows indicate the mobility of full-length
GST-Spc110p-(1-183). Lane 1, wild-type GST-Spc110p-(1-183)
(pDV29) without GST-Mps1p. Lanes 2-6: GST-Mps1p kinase
assay using as substrate wild-type GST-Spc110p-(1-183) (pDV29,
lane 2), GST-Spc110p-(1-183) containing the T64A mutation
(pJK4, lane 3), the T68A mutation (pJK2, lane 4),
the T64A,T68A double mutation (pJK7, lane 5), or the triple
S60A,T64A,T68A mutation (pJK20, lane 6).
Complementation of synthetic lethal interactions
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Fig. 7.
Mutation of phosphorylation sites in
full-length Spc110p abolishes the mitosis-specific mobility shift
in vivo. Western blot analysis using
affinity-purified anti-Spc110p antibodies and preparation of total cell
protein extracts was performed as in Fig. 1. Plasmids expressing
full-length spc110 alleles encoding the indicated mutations
of serine and threonine residues to alanine or aspartate were
transformed into yeast strain HSY2-12C using a red/white plasmid
shuffle scheme (17). Thus plasmid-encoded Spc110p is the only source of
Spc110p in HSY2-12C cells. Lane 1, plasmid pHS31
(WT). Lane 2, plasmid pJK29
(S36D,S60D,T64D,T68D). Lane 3, plasmid pJK21
(S36A,S60A,T64A,T68A). Lane 4, plasmid pJK22
(S60A,T64A,T68A). Lane 5, plasmid pJK12 (T64A,T68A).
Lane 6, plasmid pJK8 (S36A). 120- (p120) and
112-kDa (p112) Spc110p isoforms are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ken Winter, Nancy Cyrus, Julian Watts, and Reudi Aebersold for technical advice and assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health (NIH) Grant GM40506 (to T. N. D.), NIH Grant GM-51312 (to M. W.), and NIH Grant AR39730 (to K. A. R.); by the Howard Hughes Medical Institute (to N. G. A.); and by NCRR, NIH Grant P41RR11823 (to J. Y.). Mass spectra presented were generated at the University of Colorado, Boulder.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
c Supported by National Institutes of Health Grant T32-CA09437. Current address: Dept. of Cellular and Structural Biology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262.
e Supported by National Institutes of Health predoctoral fellowship GM-07135.
f Supported by a Public Health Service National Research Service Award F32-GM17946, NIGMS, NIH.
g Current address: Fred Hutchinson Cancer Research Center, 1100 E. Fairview Ave N., Seattle, WA 98109-1024.
i Current address: Dept. of Protein and Metabolite Dynamics, Novartis Agricultural Discovery Institute, 3115 Merryfield Row, Suite 100, San Diego, CA 92121.
j Current address: Dept. of Cell Biology, SR11, 10550 North Torrey Pines Rd., The Scripps Research Institute, La Jolla, CA 92037.
l To whom correspondence should be addressed: Tel.: 206-543-5345; Fax: 206-685-1792; E-mail: tdavis@u.washington.edu.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M010461200
2 D. B. N. Vinh, D. B. Friedman, and T. N. Davis, unpublished observations.
3 T. Nguyen and T. N. Davis, unpublished results.
4 E. Steiner and M. Winey, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SPB, spindle pole body; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; TLE/TLC, thin layer electrophoresis, thin layer chromatography; DTT, dithiothreitol; ESI-LC/MS, electrospray ionization, liquid chromatography mass spectrometry; ESI-LC/MS/MS, ESI liquid chromatography tandem mass spectrometry; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization, time-of-flight; endoLys-C, endoproteinase Lys-C.
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REFERENCES |
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