From the Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville Tennessee 37232
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ABSTRACT |
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Proper coordination of cytokinesis with
chromosome separation during mitosis is crucial to ensure that each
daughter cell inherits an equivalent set of chromosomes. It has been
proposed that one mechanism by which this is achieved is through
temporally regulated myosin regulatory light chain (RLC)
phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of
evidence is consistent with this model. A direct test of the importance
of RLC phosphorylation in vivo has been done only in
Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (Jordan, P., and
Karess, R. (1997) J. Cell Biol. 139, 1805-1819) but
not essential in Dictyostelium (Ostrow, B. D., Chen,
P., and Chisholm, R. L. (1994) J. Cell Biol. 127, 1945-1955). The Schizosaccharomyces pombe myosin light
chain Cdc4p is essential for cytokinesis, but it was unknown whether phosphorylation played a role in its regulation. Here we show that the
S. pombe myosin light chain Cdc4p is phosphorylated
in vivo on either serine 2 or 6 but not both. Mutation of
either or both of these sites to alanine did not effect the ability of Cdc4p to bind the type II myosin Myo2p, and cells expressing only these
mutated versions of Cdc4p grew and divided normally. Similarly, mutation of Ser-2, Ser-6, or both residues to aspartic acid did not
affect growth or division of cells. Thus we conclude that phosphorylation of Cdc4p is not essential in vivo for the
function of the protein.
Cytokinesis in diverse eukaryotes is accomplished by constriction
of an actin contractile ring. This ring is composed not only of actin
but of many other proteins including myosin, which supplies the force
needed for its constriction (1). Generally myosin consists of two heavy
chains complexed with a regulatory and an essential light chain (5). By
genetic analysis, it has been established that these conventional
myosin heavy and light chains are required for cytokinesis in
Dictyostelium discoideum (6-9) and Drosophila
melanogaster (10). It can be inferred that they are essential for
cytokinesis in most eukaryotic species, with Saccharomyces
cerevisiae being an exception. Even in this organism, however,
cell division is impaired in the absence of myosin II (11, 12).
Like many other eukaryotes, the yeast Schizosaccharomyces
pombe divides by medial fission through the use of a medially
placed actin contractile ring (13, 14). Analysis of mutants defective in medial ring formation led to the identification of a putative myosin
light chain encoded by the cdc4+ gene (4) and a
type II myosin heavy chain. Cdc4p is an essential EF hand protein that
bears significant sequence similarity to both regulatory and essential
myosin light chains from diverse eukaryotes. Cdc4p localizes diffusely
throughout the cell during interphase. However, during mitosis it
co-localizes with actin to a medial ring that constricts following
nuclear division. Myo2p, a type II myosin heavy chain, also localizes
to the medial contractile ring during mitosis, and the Myo2p ring
constricts during cell division (16, 17). Recently, it has been
established that Cdc4p directly binds Myo2p (15). Deletion of the
single IQ domain within Myo2p disrupts binding to Cdc4p (15).
Consistent with the presence of a single IQ domain within Myo2p, it
does not appear that a second light chain binds to Myo2p, and it is
likely that Cdc4p is the sole light chain for this myosin II (4).
In diverse eukaryotes, both heavy and light chains of myosin are
phosphorylated in vivo, and in vitro biochemical
experiments have shown that the phosphorylation of the regulatory light
chain regulates the actin-activated ATPase of myosin (reviewed in Ref. 5). Several phosphorylation sites have been mapped on regulatory myosin
light chains. Phosphorylation of residues at 18-21 regulates myosin
activity positively, whereas phosphorylation of more N-terminal residues (1, 2, and 9) inhibits myosin activity (reviewed in Ref. 5).
Both protein kinase C and cdc2/cyclinB kinases phosphorylate the
inhibitory N-terminal sites. It has been proposed that cdc2/cyclinB phosphorylation at these sites might serve to inhibit myosin activity at the contractile ring until nuclear division is complete (1). Both
the timing of cdc2/cyclinB inactivation at the end of anaphase and the
timing of myosin light chain phosphorylation at different sites
in vivo (18) are consistent with such a model. To determine whether phosphorylation of the single myosin light chain plays a role
in the timing of cytokinesis in fission yeast, we have examined the
phosphorylation state of Cdc4p in vivo. We have shown that
it is a phosphoprotein, and we have identified its phosphorylation sites. When these were mutated to nonphosphorylatable residues, Cdc4p
function and cell division were unaltered. Hence, the timing of
cytokinesis in fission yeast is not regulated by
phosphorylation/dephosphorylation of the myosin light chain.
Strains, Growth Media, and Genetic Methods--
The yeast
strains used in this study are listed in Table
I. Media used to grow S. pombe
cells and general genetic manipulation of S. pombe were as
described elsewhere (19). Transformations were performed by
electroporation (20). Cells were labeled with [32P]orthophosphate as detailed previously (21).
Immunoblotting and Immunoprecipitations--
S. pombe
cells were lysed in Nonidet P-40 buffer with mechanical shearing
followed by heating to 95 °C in SDS lysis buffer and dilution with
Nonidet P-40 buffer (21). Cell lysates were clarified by
centrifugation. For immunoblotting, protein extracts were resolved by
SDS-PAGE1 and transferred
electrophoretically to a polyvinylidene difluoride (PVDF) membrane
(Millipore). Blots were probed sequentially with a 1:200 dilution of
anti-Cdc4p serum (4) or a 1:1000 dilution of anti-Myo2p antibody (15)
followed by peroxidase-conjugated secondary antibody. Reactive proteins
were visualized by chemiluminescence (ECL; Amersham Pharmacia Biotech).
For immunoprecipitation of Cdc4p, lysates were prepared as above and
incubated at 4 °C for 60 min with 4 µl of antiserum. Protein
A-Sepharose was added for a further 30 min, and the immunocomplexes
were recovered by centrifugation and washed extensively.
Phosphoamino Acid Analysis and Tryptic Peptide
Mapping--
32P-Labeled Cdc4p was subjected to partial
acid hydrolysis while bound to the PVDF membrane (22), and the
phosphoamino acids were separated in two dimensions by thin-layer
electrophoresis at pH 1.9 and 3.5 (23). For tryptic digestion, pieces
of the PVDF membrane containing 32P-labeled Cdc4p were
pretreated with methanol for 30 s and then incubated at 37 °C
for 30 min with 0.1% Tween 20 in 50 mM ammonium bicarbonate, pH 8.0. After three short washes with 50 mM
ammonium bicarbonate, phosphopeptides were released from the membrane
with 2 2.5-h incubations at 37 °C in 50 mM ammonium
bicarbonate, pH 8.0, with 10 µg of
N-tosyl-L-phenylalanine chloromethyl
ketone-trypsin added for each incubation. After lyophilization, the
phosphopeptides were separated in two dimensions with electrophoresis
at pH 1.9 as detailed previously (23). Phosphoamino acids and tryptic phosphopeptides were visualized by autoradiography or with the use of a
Molecular Dynamics PhosphorImager.
In Vitro Mutagenesis--
The Ser to Ala substitutions and the
Ser to Asp substitutions were introduced by site-directed mutagenesis
into a ~2.0-kilobase cdc4+ genomic fragment in
pSK (pDM111) that had NdeI and BamHI sites inserted at the 5' and 3' ends of the coding region. The mutagenesis was performed using the Bio-Rad Muta-Gene kit according to
manufacturer's instructions. The following oligonucleotides were used:
Ser-2 to S2A, 5'-AGCATATGGCGACAGACG-3'; Ser-6 to S6A,
5'-CAGACGATGCACCTTATA-3'; Ser-2 to S2D,
5'-GAATAGTCACATATGGATACAGACGATTCACC-3'; Ser-6 to S6D,
5'-CCATTTAGAATAGTCACATATGTCGACAGACGATGATCCTTATAAACAAG-3'; and
Ser-2 Gene Replacements--
The cdc4 mutants in pIRT2 were
transformed into the heterozygous
cdc4+/cdc4::ura4+
diploid strain. Leu+ diploids were allowed to sporulate, and Leu+ Ura+
haploid progeny were isolated. These were grown in minimal media
containing uracil and leucine overnight. The following day, 3 × 107 cells were plated on minimal media containing uracil,
leucine, and 1 mg/ml 5-fluroorotic acid. Ura Cdc4p Is a Phosphoprotein--
S. pombe Cdc4p was
predicted to be a phosphoprotein based on its mobility on SDS-PAGE gels
(4). To firmly establish whether Cdc4p was indeed a phosphoprotein,
S. pombe cells were labeled with
[32P]orthophosphate, and a total cell protein lysate was
prepared. A single 32P-labeled protein of the expected size
was immunoprecipitated from the lysate by immune but not preimmune
Cdc4p serum (Fig. 1A).
Phosphoamino acid analysis of 32P-labeled Cdc4p indicated
that it was phosphorylated exclusively on serine residues (Fig.
1B).
Identification of the Cdc4p Phosphorylation
Sites--
Phosphopeptide mapping showed that Cdc4p was phosphorylated
on a single tryptic peptide (Fig. 2,
left panel). There are seven serine residues in Cdc4p
located within six tryptic peptides (Table II). Based on the predicted
electrophoretic and chromatographic mobility of the six possible
peptides, we hypothesized that peptide 1 contained the phosphorylation
site(s). This hypothesis was reinforced by the tryptic phosphopeptide
map derived from 32P-labeled HA epitope-tagged Cdc4p. This
tagged version of Cdc4p contains a single copy of the influenza
hemagglutinin HA1 epitope fused at the N terminus of Cdc4p. HA
epitope-tagged cdc4 cDNA under control of the thiamine
repressible attenuated nmt1-T4 promoter (25, 26) is fully
functional as judged by its ability to rescue growth of a
cdc4 null mutant. The tryptic phosphopeptide map of HAcdc4p
contained different phosphopeptides than the map of untagged Cdc4p
(Fig. 2, middle and right panels). This result is
explained most easily if the phosphorylation occurred on a serine(s)
within the first tryptic peptide, because the addition of the HA
epitope would lengthen the peptide and, therefore, alter its mobility (Fig. 2).
In peptide 1, there are two serine residues and hence two possible
sites of phosphorylation. To determine which serine or whether both
were phosphorylated, the cdc4+ genomic clone was
altered by site-directed mutagenesis to encode three mutant proteins
(S2A, S6A, and S2AS6A). Each of the mutants expressed episomally was
capable of rescuing both temperature-sensitive and null cdc4
mutants. To examine the phosphorylation state of the mutant
proteins, each was produced in the cdc4 null mutant, and the
cells were labeled with [32P]orthophosphate. Like
wild-type Cdc4p, the S2A and S6A mutants became labeled with
32P exclusively on serine residues (Fig.
3, A and B). In
contrast, the S2AS6A mutant protein did not become labeled detectably
with 32P (Fig. 3C). Thus, S2A and S6A represent
the phosphorylation sites of Cdc4p. Although we had no evidence that
threonine ever became phosphorylated on Cdc4p either in the wild-type
protein or in the serine substitutions, we constructed a triple mutant,
S2AT3AS6A, to ensure that phosphorylation in this region of the protein
was prevented. This triple mutant also was capable of rescuing
temperature-sensitive and null mutants of cdc4. In fact, the
physiological experiments described below were performed with a mutant
strain in which the wild-type copy of cdc4+ was
replaced with the cDNA encoding S2AT3AS6A. In this strain, the
triple mutant protein was produced at levels equivalent to wild-type
Cdc4p and was not phosphorylated (Fig.
4).
Cdc4p Phosphorylation Is Not Required for Its Function--
As
mentioned above, we were able to construct a gene replacement strain in
which cdc4+ was replaced with a construct
encoding S2AT3AS6A. A priori, we could conclude that the
phosphorylation of Cdc4p was not required for its essential function.
This strain not only produced wild-type levels of the mutant protein,
but its doubling time, growth on different media, and growth on media
containing the microtubule-destabilizing drug, thiabendazole, were all
indistinguishable from that of wild-type cells (data not shown). We
also tested whether the binding of Cdc4p to Myo2p was affected by the
lack of phosphorylation. The same level of Myo2p co-immunoprecipitated
with mutant Cdc4p as with wild-type Cdc4p (Fig.
5A).
Because we did not detect an alteration in cell growth or division when
Cdc4p was not phosphorylated, we considered the possibility that
dephosphorylation rather than phosphorylation of Cdc4p might serve some
essential regulatory role. To test this potentiality, we altered the
cdc4+ cDNA by site-directed mutagenesis to
encode proteins in which the serine phosphorylation sites were replaced
with aspartic acid residues (S2D, S6D, and S2D/S6D). In some instances,
the negative charge of aspartic acid can mimic, at least partially, the
consequence of phosphorylation. Each aspartic acid mutant was assayed
for its ability to complement both temperature-sensitive and null cdc4 mutants when expressed from the repressed
nmt1 promoter. All three mutants were able to do so (data
not shown). To exclude the possibility that multiple copies of these
genes might obscure a phenotype, they were each integrated into the
genome in single copy by replacing the cdc4 null mutation.
All three mutant strains were viable and grew with wild-type kinetics
on plates (Fig. 6) and in liquid medium
(data not shown). Additionally, they were not sensitive to changes in
media composition, temperature, or the microtubule-destabilizing drug,
thiabendazole (data not shown).
Regulatory Light Chain (RLC) Phosphorylation and Regulation of
Cytokinesis--
Numerous previous biochemical studies have indicated
that myosin RLC phosphorylation may play a key role in regulating
myosin function (for review see Ref. 27). Phosphorylation by myosin light chain kinase has been shown to increase the actin-activated ATPase activity of myosin II. Although myosin light chain kinase phosphorylation of the RLC (on serine 19 in vertebrate cells) is also
required for assembly of vertebrate smooth muscle and nonmuscle myosins
into filaments (28), phosphorylation of the Dictyostelium
RLC does not seem to be required for filament assembly (5, 29).
Additionally the vertebrate RLC has been shown to be phos-phorylated
in vivo on serine 1 and 2 (18). These sites have been shown
to be phosphorylated in vitro by both Cdc2 and protein
kinase C, which results in inhibition of the actin activated ATPase
activity of myosin (see Ref. 1 and references therein). These results
led Satterwhite and Pollard (1) to propose an elegant model where,
early in mitosis, phosphorylation by CDC2 would inhibit assembly of
active myosin at the cortex, then inactivation of CDC2 would allow
dephosphorylation of these sites, and subsequent phosphorylation by
myosin light chain kinase would trigger myosin assembly and cleavage
furrow formation. This model was further supported by a study showing
that Ser-1 and Ser-2 phosphorylation was maximal in mitotic cells, and
then during cytokinesis Ser-1 and Ser-2 phosphorylation decreased, and
Ser-19 phosphorylation increased (18). These results have been further
supported by cytological examination of cells proceeding through
mitosis using Ser-19 phosphoepitope-specific antibodies (30).
Although the experiments described above make a compelling case for the
importance of RLC phosphorylation in the regulation of cytokinesis, the
results are only correlative and do not demonstrate whether these
events are essential in vivo. A direct test of the importance of these phosphorylation events in vivo using
genetic systems has provided mixed results. In
Dictyostelium, RLC null cells are unable do undergo
cytokinesis in liquid culture (8) just like cells that are null for the
myosin heavy chain (6, 7). However, mutant RLCs that have had the
activating phosphorylation site (Ser-13) mutated to alanine are able to
fully complement the defects of the RLC null alleles even though the
myosin isolated from these cells displayed reduced actin-activated
ATPase activity (3). These experiments argue that although the RLC
phosphorylation is conserved and may be important for the long term
fitness of the organism, it is not essential. In contrast, experiments
in Drosophila have shown that RLCs with alanine substitution
mutations in the activating phosphorylation sites are unable to rescue
RLC null alleles (2). One explanation for these different results may
that in Dictyostelium, RLC phosphorylation only modulates the actin-activated ATPase activity of myosin, whereas in
Drosophila, both activity and assembly are affected. To date
no genetic studies have investigated the importance of the RLC
inhibitory phosphorylation sites in vivo.
Myosin Light Phosphorylation in S. pombe--
As has been observed
in other systems, type II myosin and its associated light chain play
essential roles in fission yeast cytokinesis (4, 16, 17, 31-33). In
the studies presented here, we demonstrated that like vertebrate RLCs,
Cdc4p is phosphorylated on two sites near the N terminus of the protein
(Ser-2 and Ser-6). Also like vertebrate cells, we do not observe both
phosphorylation events at once. Cells seem to have one site or the
other phosphorylated, but not both, suggesting that the phosphorylation
of Cdc4p is regulated and that perhaps phosphorylation of one site
inhibits phosphorylation of the other. The fact that the
phosphorylation site at Ser-6 is a consensus Cdc2p phosphorylation site
further suggests that Cdc4p phosphorylation may be cell
cycle-regulated. Unfortunately, we were unable to determine whether
either of these sites was phosphorylated in a cell cycle-specific
manner, and we did not observe any changes in the total phosphorylation
levels of the protein throughout the cell cycle (data not shown). The lack of a phenotype when these sites are mutated either singly or in
combination shows that these sites are not essential for cells to carry
out cytokinesis and that yeast myosin II may function more like the
Dictyostelium myosin. It is also possible that, like in
Dictyostelium, heavy chain phosphorylation may be important for regulation of myosin function, because we have observed that Myo2p
is a phosphoprotein in
vivo.2 It will be
important in the future to develop procedures for purifying yeast
myosin so that biochemical effects of the phosphorylation site
mutations can be determined, as has been done in
Dictyostelium.
Some caution should be used in drawing the comparison between S. pombe myosin II and myosin II from Dictyostelium and
vertebrates. The two type II myosin genes that have been identified in
S. pombe have both been termed unconventional type II
myosins largely because the tails have numerous proline residues that
could potentially disrupt coiled coil-mediated oligomerization (for
review see Ref. 34). Furthermore, a recent study showed that Myo2p
contains a single functional IQ site that binds Cdc4p, and thus, Cdc4p may be the only light chain for this myosin (15). At present it is
unclear whether these myosins represent primordial type II myosins, the
first members of a new family, or diverged yeast forms of myosin II.
For this reason, regulatory effects of phosphorylation may be somewhat
different from other myosins. Still, it is striking that the similar
pattern of two N-terminal phosphorylation sites is conserved, and the
simple fact that this phosphorylation has been maintained throughout
evolution indicates it that must be doing something beneficial for the
cell, which may not be readily observed under our laboratory growth
conditions. If Cdc4p phosphorylation serves a somewhat redundant
function, as our results suggest, it may be of future interest to look
for mutations that are synthetically lethal in combination with Cdc4p
phosphorylation site mutants, to identify genes that are
essential in the absence of proper regulation of Cdc4p by phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. pombe strains used in this study
Ser-6 to S2D/S6D,
5'-CCATTTAGAATAGTCACATATGGATACAGACGATGATCCTTATAAACAAG-3'. The S2A/S6A
and S2A/T3A/S6A mutations were made by polymerase chain reaction with
using the S6A clone as a template followed by cloning into pDM111. The
5' oligonucleotides used were 5'-GGCATATGGCGGCAGACGATGCACCT-3' and
5'-GGCATATGGCGACAGACGATGCA-3' respectively, and the 3' oligonucleotide was cdc4stp (4). The gene fragment was removed from pDM111 using
XbaI and Acc651 and subcloned into the yeast expression vector pIRT2 (24).
Leu
colonies were then
selected. To confirm that the correct gene replacement was present
within these cells, genomic DNA was prepared from the colonies, and the relevant piece of cdc4 was amplified by the polymerase chain
reaction using the oligonucleotides cdc4seq1
(5'-GACCAGTTCCATGGCGGTC-3') and cdc4PCR1 (5'-GTAAATTGAAGGTTGAGCG-3').
The polymerase chain reaction product was sequenced directly using the
cdc4seq1 oligonucleotide.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cdc4p is a phosphoprotein. A,
wild-type 972 cells were labeled with [32P]orthophosphate
and lysed in SDS lysis buffer. Anti-Cdc4p serum (I) was
added to one-half of the lysate, and preimmune (PI) serum
was added to the other half. The immunoprecipitates were resolved by
SDS-PAGE and transferred to a PVDF membrane. Labeled proteins were
detected by autoradiography. The position of the band corresponding to
Cdc4p is indicated with an arrow. B, the piece of
PVDF membrane containing Cdc4p was analyzed for its phosphoamino acid
content. The positions of the phosphothreonine (T) and
phosphotyrosine (Y) standards are diagrammed. S,
phosphoserine.
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Fig. 2.
Two-dimensional tryptic phosphopeptide maps
of Cdc4p. 32P-Labeled Cdc4p obtained from wild-type
cells as in Fig. 1 and 32P-labeled HAcdc4p produced in
cdc4 null cells were isolated in parallel and digested with
trypsin. The tryptic phosphopeptides were separated in two dimensions
as described under "Experimental Procedures." Electrophoresis was
performed in the horizontal dimension at pH 1.9, with the anode on the
left. In each case the origin is marked with arrows. In the
right panel, tryptic peptides from Cdc4p were analyzed
together with tryptic peptides from HAcdc4p.
Cdc4p serine-containing tryptic peptides
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Fig. 3.
Identification of the Cdc4p phosphorylation
sites. A, the cdc4-S2A and cdc4-S6A proteins were
produced in cdc4 null cells, and the cells were labeled with
[32P]orthophosphate. Total protein lysates were prepared
from the cells, and the Cdc4 proteins were immunoprecipitated, resolved
by SDS-PAGE, transferred to a PVDF membrane, and detected by
autoradiography. The arrow indicates the band corresponding
to Cdc4p. B, phosphoamino acid content of the bands detected
in panel A. The positions of the phosphothreonine
(T) and phosphotyrosine (Y) standards are
diagrammed. S, phosphoserine. C, wild-type cdc4
and cdc4-S2AS6A protein were produced in cdc4 null cells,
and the cells were labeled with [32P]orthophosphate.
Total protein lysates were prepared from the cells, and the cdc4
proteins were immunoprecipitated, resolved by SDS-PAGE, and transferred
to a PVDF membrane. Labeled proteins were detected by autoradiography.
The arrow indicates the position of Cdc4p.
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Fig. 4.
The S2AT3AS6A protein is not
phosphorylated and is functional. A strain was constructed in
which sequences encoding the S2AT3AS6A mutant replaced the wild-type
cdc4+ coding region (see "Experimental
Procedures"). Cdc4p was immunoprecipitated from a wild-type strain
and from the S2AT3AS6A strain that were either unlabeled (A)
or labeled with [32P]orthophosphate (B). The
immunoprecipitates were resolved by SDS-PAGE and transferred to a PVDF
membrane. Cdc4 proteins from the unlabeled strains were detected by
immunoblotting with anti-Cdc4p serum (A). Labeled
immunoprecipitated proteins were detected by autoradiography
(B).
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Fig. 5.
Phosphorylation is not required for Myo2p
interaction. Lysates prepared from wild-type (lanes 1 and 2) or the S2A/T3A/S6A strain (lanes 3 and
4) were subject to immunoprecipitation with either preimmune
(lanes 1 and 3) or immune (lanes 2 and
4) Cdc4p serum. Immunoprecipitates were resolved by SDS-PAGE
and transferred to a PVDF membrane, and the membrane was cut in half.
The top half was probed with anti-Myo2p serum (A), and the
bottom half was probed with anti-Cdc4p serum (B). Proteins
were visualized by enhanced chemiluminescence (ECL). The
arrows indicate the positions of Myo2p in (A) and
Cdc4p in (B).
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Fig. 6.
Growth of cdc4 phosphorylation mutants.
Strains in which the cdc4+ gene had been
replaced with the indicated mutants were streaked to yeast-glucose
plates, and colonies were allowed to grow for 3 days at 32 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. M. Balasubramanian for critical reading of the manuscript and for antibodies to Myo2p.
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FOOTNOTES |
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* This work was supported by the Howard Hughes Medical Institute of which K. L. G. is an Associate Investigator.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.
Supported by National Institutes of Health post-doctoral training
Grant GM16145. Current address: Dept. of Molecular Genetics and
Microbiology, University of Massachusetts Medical Center, Worcester, MA 01605.
§ To whom correspondence should be addressed. Tel.: 615-343-9502; Fax: 615-343-0723; E-mail:kathy.gould{at}mcmail.vanderbilt.edu.
2 K. Gould, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HA, hemagglutinin; RLC, regulatory light chain.
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