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Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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To identify septin-interacting proteins in
Saccharomyces cerevisiae, we screened for mutations
that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and
Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with
the COOH-terminal portion of the Cdc3p septin, and
Gin4p colocalized with the septins at the mother-bud
neck. This localization depended on the septins and on
the COOH-terminal (nonkinase) region of Gin4p, and
overproduction of this COOH-terminal region led to a
loss of septin organization and associated morphogenetic defects. We detected no effect of deleting
YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led
to a striking reorganization of the septins accompanied
by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared
to occur efficiently. Two other proteins that localize to
the neck in a septin-dependent manner showed similar
reorganizations and also appeared to remain largely
functional. The septin organization observed in gin4
vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The
organization of the septins observed in gin4 cells and
in cells responding to pheromone appears to support
some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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THE septins are a family of proteins that were first
identified in the budding yeast Saccharomyces cerevisiae but have since been identified also in a variety
of other fungi and animals (for review see Cooper and
Kiehart, 1996
; Longtine et al., 1996; Longtine and Pringle,
1998). The septins localize to the cleavage site and are essential for cytokinesis in both yeast and animal cells, but
their precise role(s) in this process are unknown. In addition, it appears that the septins also have roles in processes
other than cytokinesis. For example, in Drosophila, septins are highly concentrated in the basolateral surfaces of
ovarian follicle cells and in neurons of the embryonic central nervous system (Neufeld and Rubin, 1994; Fares et al.,
1995); there are as yet no good clues to the roles of the
septins in these polarized, nondividing cells. In S. cerevisiae, two of the seven septins are expressed only in sporulating cells and appear (from protein-localization data and
mutant phenotypes) to be involved in the poorly understood process by which the meiotic nuclei are packaged
into spores (De Virgilio et al., 1996; Fares et al., 1996).
At least some septin functions appear to involve a role
in organizing other proteins at the cell surface. For example, the septins are involved in the selection of axial budding sites in haploid cells (Flescher et al., 1993; Chant et al.,
1995); this function reflects the role of the septins in the localization of Bud3p and Bud4p, two proteins that are constituents of a transient mark at the division site that serves
to direct the next budding event to an adjacent location
(Chant et al., 1995; Sanders and Herskowitz, 1996). In addition, the septins are necessary for the proper localization
of Chs3p (the catalytic subunit of chitin synthase III), and
hence for the normal localization of chitin deposition to a
ring at the base of the bud (DeMarini et al., 1997). Localization of Chs3p involves at least two other proteins, Chs4p, which appears to interact directly with Chs3p, and
Bni4p, which appears to interact directly both with Chs4p
and with one of the septins.
Another interesting class of questions about the septins
concerns the relationship between the function of these
proteins and their assembly into higher-order structures.
In S. cerevisiae, the vegetatively expressed septins appear
to be constituents of a set of filaments found on the cytoplasmic face of the plasma membrane in the mother-bud
neck (Byers and Goetsch, 1976; Byers, 1981; Longtine et al.,
1996; Frazier et al., 1998). However, the relationship between the function of the septins and their assembly into
these filaments remains obscure, as do the mechanisms
controlling the assembly and disassembly of the filaments.
In addition, although some progress has been made in analyzing septin complexes (including short filaments) isolated from Drosophila (Field et al., 1996), it is not yet clear
whether the septins of Drosophila or other organisms are
organized in vivo into filaments like those seen in S. cerevisiae.
In this paper, we report the results of a genetic screen in S. cerevisiae that identified a protein kinase, Gin4p, that appears to coassemble with the septins at the mother-bud neck and play an important role in their assembly. Interestingly, analysis of gin4 mutant strains suggests that the septins and septin-associated proteins can remain largely functional after a seemingly drastic change in their organization.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Strains, Growth Conditions, and Genetic and DNA Methods
The S. cerevisiae strains used in this study are described in Table I. Yeast
media (YM-P rich liquid medium, YPD rich solid medium, synthetic minimal [SD]1 medium, synthetic complete [SC] medium lacking specific nutrients, and sporulation medium) have been described previously (Lillie and
Pringle, 1980; Guthrie and Fink, 1991). S. cerevisiae strains were grown at
30°C except where noted. Cultures were treated with 
mating pheromone
(Sigma Chemical Co., St. Louis, MO) as described by Evangelista et al.
(1997). Escherichia coli strain DH12S (Life Technologies, Gaithersburg,
MD) and standard media and methods (Ausubel et al., 1995) were used
for plasmid manipulations. Transformation of yeast was performed as described by Gietz et al. (1992), and other yeast genetic manipulations used
standard procedures (Guthrie and Fink, 1991).
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Standard methods (Ausubel et al., 1995) were used for DNA manipulations except where noted. DNA-DNA blot hybridization was performed
as described previously (Ausubel et al., 1995) and site-specific mutagenesis was carried out using the pALTER system as described by the manufacturer (Promega, Madison, WI). The oligonucleotides used in this study
are described in Table III and were obtained from Integrated DNA Technologies (Coralville, IA). Vent DNA polymerase (New England Biolabs,
Beverly, MA) was used for the PCR synthesis of DNA fragments for cloning; Taq DNA polymerase (Promega) was used in other PCR applications. Primer pairs ML38 and ML39 and ML40 and ML41 were used to
generate DNA cassettes for replacement of GIN4 and YCL024W with
TRP1 and HIS3, respectively, using the PCR method of Baudin et al.
(1993) and plasmids pRS304 and pRS303 (Sikorski and Hieter, 1989) as
templates. The success of the gene replacements was verified by PCR using yeast genomic DNAs as templates, primers ML38 and MLP54-5 for
GIN4, and two different pairs of primers (ML18 and ML41; ML195 and
ML196) for YCL024W.
Plasmid Constructions
Most plasmids used in this study are listed in Table II; others are described where appropriate below. YCpGALGST was constructed by inserting an ~1.8-kb StuI/HindIII fragment containing the GAL1/10 promoter from pEG(KT) (Mitchell et al., 1993) into SmaI/HindIII-digested YCplac111. YCp/CDC12 was constructed by subcloning an ~2-kb XbaI/ PstI fragment containing CDC12 from YEp24(CDC12)N into XbaI/PstI-digested YCplac111. p54-6 was a primary isolate from the plasmid library that contained MPK1/SLT2, and p54-15 was a primary isolate from the
plasmid library that contained GIN4 (see below). YCp/GIN4, YEp/GIN4,
and pALTER/GIN4 were constructed by subcloning an ~7-kb XbaI
fragment from p54-15 into XbaI-digested YCplac111, YEplac181, and
pALTER, respectively.
To construct plasmids containing tagged or mutated alleles of GIN4,
single-stranded DNA from plasmid pALTER/GIN4 was used with primer
ML62 (which mutates the lysine codon at position 48 to a methionine
codon), primer ML63 (which introduces a NotI site just upstream of the
GIN4 stop codon), or both, to create plasmids pALTER/gin4K48M, pALTER/
GIN4NotI, and pALTER/gin4K48MNotI. NotI fragments containing sequences encoding either a triple hemagglutinin tag (3HA) (Tyers et al.,
1993) or glutathione-S-transferase (GST) (provided by D. DeMarini, University of North Carolina, Chapel Hill, NC) were cloned into NotI-digested
pALTER/GIN4NotI and pALTER/gin4K48MNotI to yield plasmids pALTER/GIN4-3HA, pALTER/GIN4-GST, and pALTER/gin4K48M-3HA.
The appropriate XbaI fragments from the pALTER derivatives were then
cloned into XbaI-digested YCplac111, YEplac181, or YIplac204 to yield
plasmids YCp/gin4K48M, YEp/gin4K48M, YCp/GIN4-3HA, YCp/gin4K48M-3HA, and YIp/GIN4-GST. Before transformation into yeast, YIp/GIN4-GST was digested with ApaI to target integration to the ura3 locus.
To construct plasmid YCpGALGST/GIN4N, which contains the kinase region of GIN4 (codons 1-315) fused to GST-encoding sequences under the control of the GAL1/10 promoter, the PCR product obtained using primers ML67 and ML73 and pALTER/GIN4 as template was digested with BglII and XhoI and cloned into BamHI/SalI-digested YCpGALGST. The GIN4 ORF in YCpGALGST/GIN4N includes eight COOH-terminal non-GIN4-encoded amino acids (LDSSSSLA) followed by a stop codon. To construct plasmid YCpGALGST/GIN4C, which contains the COOH-terminal nonkinase region of GIN4 (codons 295-1,142) fused to GST-encoding sequences under the control of the GAL1/10 promoter, an ~2.2-kb SalI fragment from pEG202/GIN4C (see below) was cloned into SalI-digested YCpGALGST.
To construct plasmids for two-hybrid analyses, the kinase (codons 1-294) and nonkinase (codons 295-1,142) regions of GIN4 were amplified by PCR using primers ML67 and ML72 and ML69 and ML70, respectively, and plasmid pALTER/GIN4 as template. The products were digested with EcoRI and XhoI or with NcoI and XhoI, respectively, and cloned into EcoRI/XhoI- or NcoI/XhoI-digested pEG202 to yield plasmids pEG202/GIN4N and pEG202/GIN4C, respectively. pEG202/ GIN4NFL, which contains nearly full-length GIN4 (codon 18 to past the stop codon), was constructed by cloning an NcoI-XhoI fragment from plasmid p54-15 into NcoI/XhoI-digested pEG202.
Cloning and Sequencing of GIN4
A plasmid shuffle was performed in mutant isolate JFY54A (see Results) to replace plasmid pB12G with the URA3-marked plasmid YEp24 (CDC12)N, creating strain M-100. M-100 was then transformed with a library of S. cerevisiae genomic DNA fragments in the low-copy plasmid YCp50-LEU2 (provided by P. Hieter, University of British Columbia, Vancouver, British Columbia, Canada), and ~9,000 transformants were replica-plated at 23°C to SC-Leu medium containing 0.1% 5-fluoroorotic acid (5-FOA) to select against cells containing plasmid YEp24(CDC12)N. Nine clones were able to grow, indicating that they contained plasmids that rescued the synthetic lethal phenotype. Six of these clones were also able to grow at 37°C and thus were presumed to harbor CDC12-containing plasmids. Plasmids from the remaining three clones (p54-4, p54-6, and p54-15) were recovered into E. coli and confirmed to rescue the 5-FOA sensitivity of strain M-100. Restriction enzyme and DNA-DNA blot hybridization analyses indicated that plasmids p54-4 and p54-15 contained identical ~12-kb inserts of genomic DNA (data not shown); thus, only p54-15 was used in further studies.
An ~7-kb XbaI fragment from p54-15 was ligated in both orientations
into XbaI-digested pRS315 (Sikorski and Hieter, 1989); both of the resulting plasmids (p54-110 and p54-170) rescued the 5-FOA sensitivity of
strain M-100. Plasmid pSacI was created by digesting p54-110 with SacI,
which cuts once in GIN4 and once in the plasmid polylinker, and then religating. Other deletions in the XbaI inserts of plasmids p54-110 and p54-170 were made using an exonuclease III/mung-bean nuclease deletion
method as described previously (Ausubel et al., 1995), with ApaI used as the blocker enzyme and HindIII used to allow digestion by exonuclease III. The resulting deletion plasmids (including p110-1, p110-3, p170-20, and p170-10; see Fig. 1 A) were assayed for the ability to confer
5-FOA resistance to strain M-100 and were used as substrates for double-stranded DNA sequencing using T3 or T7 primers (Sikorski and Hieter,
1989) (Stratagene, La Jolla, CA) or custom primers where necessary to
complete the DNA sequence. Approximately 4 kb were sequenced (from
109 nucleotides upstream of the GIN4 start codon to 454 nucleotides
downstream of the stop codon); the sequence was identical to that subsequently released by the genome project.
Two-hybrid Analysis
Two-hybrid analyses (Fields and Sternglanz, 1994) were conducted using
the system described by Brent and coworkers (Gyuris et al., 1993;
Ausubel et al., 1995). Strain EGY48J was cotransformed with DNA-binding domain (DBD) and activation domain (AD) plasmids and transformants were selected on solid SD + Leu medium. Quantitative assays for
-galactosidase activity were then performed in triplicate as described by
De Virgilio et al. (1996). In brief, at least three independent transformants
were grown for 16 h at 30°C in SD + Leu medium containing 1% raffinose
and 2% galactose instead of glucose, and the levels of -galactosidase activity were determined. pJG4-5PL-derived plasmids encoding AD fusions
of full-length Cdc10p and full-length, NH2-terminal, and COOH-terminal
regions of Cdc3p, Cdc11p, and Cdc12p have been described previously
(De Virgilio et al., 1996).
Antibodies, Protein Procedures, and Microscopy
To prepare Gin4p-specific antibodies, the COOH-terminal region of
GIN4 from codon 936 to 16 nucleotides past the stop codon was amplified
by PCR using primers ML102 and ML103 and pALTER/GIN4 as template. The PCR product was digested with BamHI and EcoRI or with
BamHI and HindIII and ligated into BamHI/EcoRI-digested pMAL-C2
(New England Biolabs) or BamHI/HindIII-digested pGEX-2T (Pharmacia Biotech, Piscataway, NJ) to create plasmids pMAL-C2/GIN4C and
pGEX-2T/GIN4C, respectively. Fusion proteins were expressed from
these plasmids and purified by batch methods according to standard protocols (Ausubel et al., 1995), then injected into rabbits using standard procedures (Cocalico Biologicals, Reamstown, PA). The antibodies raised
against GST-Gin4p were then affinity purified using MalE-Gin4p on nitrocellulose blots (Pringle et al., 1991). Antibodies to Cdc3p, to Cdc11p, to
Bni4p, and to Bud4p have been described previously (Ford and Pringle,
1991; Kim et al., 1991; Sanders and Herskowitz, 1996; DeMarini et al.,
1997). Polyclonal antibodies to GST (Pharmacia Biotech) were purified as
described by Bi and Pringle (1996). Monoclonal anti-HA antibody (HA.11) and monoclonal anti-tubulin antibody (YOL1/34) were purchased from Berkeley Antibody Co. (Richmond, CA) and Accurate
Chemical and Scientific Company (Westbury, NY), respectively. FITC- or rhodamine-labeled secondary antibodies and rhodamine-phalloidin were
purchased from Jackson ImmunoResearch Laboratories (West Grove,
PA) and Molecular Probes (Eugene, OR), respectively.
Yeast proteins were isolated for Western blot analysis by resuspending
whole cells in 2× Laemmli sample buffer (Laemmli, 1970) and boiling for
5 min. Western blot analysis was performed as described by Ausubel et al.
(1995), using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL) for detection.
Differential interference contrast (DIC) and fluorescence microscopy
were performed using a Nikon Microphot SA microscope (Tokyo, Japan).
Cells were stained with Calcofluor to visualize chitin, stained with rhodamine-phalloidin to visualize F-actin, or prepared for immunofluorescence
as described previously (Pringle et al., 1989, 1991), using bisBenzamide
(Sigma Chemical Co.) in the mounting medium to stain DNA. To determine
whether the cells in clumps had completed cytokinesis, cells were either sonicated or fixed and then treated with cell wall-digesting enzymes without sonication (Pringle and Mor, 1975). To evaluate axial or nonaxial budding, 
200
Calcofluor-stained cells with three or more bud scars were examined; cells
were scored as budding axially if all bud scars were in a single chain.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Identification of MPK1/SLT2 and GIN4 by Synthetic Lethal Interaction with a cdc12 Septin Mutation
To identify proteins that interact with the S. cerevisiae septins, we screened for mutations that are lethal in combination with a cdc12-5 temperature-sensitive mutation. This
screen identified several genes including BNI1 and CHS4
(DeMarini et al., 1997).2 Another complementation group,
defined by strain JFY54A, displayed a segregation pattern
suggesting that two mutations were required for synthetic
lethality with cdc12-5 at 23°C. To identify the corresponding genes, a library was screened for plasmids that could
rescue the synthetic lethality of strain M-100 (a derivative of JFY54A), yielding plasmids p54-6 and p54-15 (see Materials and Methods). Cells of strain M-100 containing p54-15 in place of the CDC12 plasmid grew at a rate comparable to that of strain M-100 itself, but cells containing only
p54-6 grew more slowly.
Deletion and partial sequence analysis of p54-6 (data
not shown) indicated that the complementing open reading frame (ORF) was MPK1/SLT2, which encodes one of
several S. cerevisiae MAP kinase homologues (for review
see Levin and Errede, 1995); mutation of MPK1 results in
temperature-dependent osmotic sensitivity (Torres et al.,
1991; Lee et al., 1993). A deletion of MPK1, in the absence of other known mutations, was synthetically lethal with
the cdc12-6 temperature-sensitive allele at 30°C; that is, in
23 tetrads dissected from a cross of strains M-238 and
DL454 (Table I), no segregants were recovered that were
Trp+ (indicating the presence of mpk1) and unable to
grow at 37°C on medium containing 1 M sorbitol (indicating the presence of cdc12-6), yet able to grow at 30°C on
medium lacking sorbitol (a condition normally permissive
for both cdc12-6 and mpk1 single-mutant strains). Low-copy plasmids containing either CDC12 (YCp/CDC12) or
MPK1 (YCp50[MPK1]) could rescue the synthetic lethality at 30°C. In the absence of a rescuing plasmid, cdc12-6
mpk1 double-mutant cells incubated at 30°C underwent
several cell divisions before arresting growth with cells of
nearly normal morphology that appeared to be in various
stages of the cell cycle (data not shown).
An ~3.6-kb region of p54-15 responsible for rescue of
the synthetic lethality of strain M-100 was identified by
subcloning and deletion analysis (Fig. 1 A). Sequencing of
this region revealed a single long ORF encoding a predicted protein of 1,142 amino acids (129,778 kD). The sequence of codons 497-535 of this ORF had been deposited
previously in GenBank; the identified ORF was named
GIN4 because it was isolated in a screen for sequences
whose overexpression resulted in a growth inhibitory phenotype (Akada et al., 1997). Subsequent release of the sequence of chromosome IV by the Saccharomyces genome
project identified GIN4 as ORF YDR507C.
Homology of Gin4p to Protein Kinases
Analysis of the predicted Gin4p sequence revealed that it
contains an NH2-terminal protein kinase domain and a
long COOH-terminal nonkinase region (Fig. 2 A), and
Gin4p has been shown to have protein kinase activity in
vitro (Altman and Kellogg, 1997). The nonkinase region
contains no obvious motifs except for a region predicted
(Lupas, 1996) to form a coiled coil (Fig. 2 A). Gin4p is
most similar to the protein encoded by S. cerevisiae ORF
YCL024W, identified by the genome project; Ycl024Wp
displays 45% overall identity to Gin4p, with 76% identity
in the kinase domain and 35% identity in the nonkinase
region (Fig. 2 A). Although the Gin4p and Ycl024Wp nonkinase regions are not significantly homologous to any
other proteins currently in the databases, their kinase domains are particularly similar to the corresponding domains of four other kinases, S. cerevisiae Hsl1p/Nik1p
(62% identity), Schizosaccharomyces pombe Nim1p/
Cdr1p (49% identity) and Cdr2p (52% identity), and a
Caenorhabditis elegans kinase identified during genomic
sequencing (51% identity) (Fig. 2 B). In addition, phylogenetic analysis (performed by S. Hanks, Vanderbilt University, Nashville, TN) suggests that Gin4p, Ycl024Wp,
Hsl1p/Nik1p, Nim1p/Cdr1p, Cdr2p, and (less closely) the
C. elegans kinase constitute a distinct family (Fig. 2 B).
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Deletion of GIN4 and Genetic Interactions between GIN4 and CDC12
The GIN4 ORF was precisely replaced with TRP1 as described in Materials and Methods (Fig. 1, A and B). Tetrads from strain M-261 (GIN4/gin4-9) segregated 2+:2
for TRP1 (gin4-9) and 4+:0 for viability at temperatures
from 18° to 39°C, indicating that GIN4 is not essential for
viability.
Because of the complicated genetics of the original synthetic lethal mutant, we asked if gin4-9 and cdc12 mutations were synthetically lethal in the absence of other
known mutations. Strains M-238 (cdc12-6 GIN4) and
M-267 (CDC12 gin4-9) were crossed, and 24 tetrads
were dissected onto YPD plates containing 1 M sorbitol at
23°C. 94 out of 96 spores were viable. The segregants were scored for gin4-9 (Trp+) and cdc12-6 (inviability at 37°C)
and streaked on YPD plates to assay viability at 30°C. As
expected, all 22 wild-type segregants and all 25 gin4-9
single-mutant segregants were viable at 30°C, and cdc12-6
single-mutant segregants were mostly viable at this temperature (22 viable out of 24 total). In contrast, all 23 cdc12-6 gin4-9 double-mutant segregants were inviable
at 30°C. The synthetic lethality was confirmed by showing
that a low-copy plasmid containing either GIN4 or
CDC12, but not a control plasmid, could rescue the viability of cdc12-6 gin4-9 segregants at 30°C (Fig. 1 C). cdc12-6
gin4-9 double-mutant strains grew slowly even at 23°C (Fig. 1 C) and displayed a morphology very similar to that
of a cdc12-6 single-mutant strain at restrictive temperature
(Fig. 1 D) (Hartwell, 1971; Adams and Pringle, 1984); as
expected from their abnormal morphology, the cdc12-6
gin4-9 cells grown at 23°C also lacked detectable septin
localization to the mother-bud necks (data not shown). A
low-copy plasmid containing either GIN4 or CDC12 restored both normal cell morphology (Fig. 1, E and F) and
septin localization to the necks (data not shown). These
data suggest that deletion of GIN4 exacerbates the functional defect of the mutant Cdc12p and hence that Gin4p
normally has a positive role in septin function.
To test this hypothesis further, we asked if overexpression of GIN4 could rescue the temperature-sensitive lethality of a cdc12-6 strain. As expected, cells of strain M-238 containing a control plasmid were viable at 23°C but inviable at 32° or 37°C, and cells containing a CDC12 plasmid were viable at all three temperatures (Fig. 3 A). In contrast, cells containing either a low-copy or a high-copy GIN4 plasmid were able to grow at 32°C although not at 37°C (Fig. 3 A), and the low-copy GIN4 plasmid even restored nearly normal morphology to the cells grown at 32°C (Fig. 3, B-D). (Because a high-copy GIN4 plasmid itself causes morphological defects [see below], it could not be tested for rescue of the cdc12-6 morphological defect.)
To ask if the protein kinase activity of Gin4p is necessary for its positive role in septin function, we tested the
suppression ability of the gin4K48M and gin4K48A alleles, in
which an invariant lysine of the kinase domain is altered
(Fig. 2 A). Mutation of this lysine eliminates or greatly reduces the catalytic ability of a variety of kinases (for review see Bossemeyer, 1995; Hanks and Hunter, 1995; Taylor et al., 1995), and Gin4pK48A has been shown to have
little or no kinase activity in vitro (Altman and Kellogg,
1997). In contrast to the corresponding GIN4 plasmids, a
low-copy gin4K48M plasmid, a low-copy gin4K48A plasmid,
and a high-copy gin4K48M plasmid all failed to rescue either
the synthetic lethality of a cdc12-6 gin4-9 strain or the viability of a cdc12-6 strain at 32°C (Fig. 3 A, and data not
shown). Moreover, low-copy gin4K48M and gin4K48A plasmids also failed to rescue the morphology of cdc12-6 cells (strain M-238) grown at 32°C (data not shown). The lack
of rescue by gin4K48M and gin4K48A was not due to a lack of
protein, as these alleles are expressed at levels similar to
those of wild-type Gin4p (Fig. 4, lanes 4 and 6) (Altman
and Kellogg, 1997). Thus, the positive role of Gin4p in septin function appears to involve its kinase activity (see also
below).
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Two-hybrid Interaction between Gin4p and Cdc3p
Gin4p might affect septin function directly or indirectly. As one approach to this question, we tested for Gin4p- septin interactions using the two-hybrid system. Interaction was detected between the Gin4p kinase domain and either full-length Cdc3p or a fragment comprising the 101 COOH-terminal amino acids of Cdc3p (Table IV). Other interactions were not detected; in particular, a construct containing all but the first 17 codons of Gin4p did not show a detectable interaction with Cdc3p (Table IV; see Discussion). Thus, Gin4p may influence septin function, at least in part, by a direct interaction between its kinase domain and the COOH-terminal portion of Cdc3p.
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Localization of Gin4p in Wild-type and Septin-mutant Strains
To explore further the apparent Gin4p-septin interaction,
we investigated the intracellular localization of Gin4p using three approaches. First, we raised and affinity purified
antibodies against the COOH-terminal portion of Gin4p
(see Materials and Methods). In extracts of wild-type cells,
the purified antibodies recognized a polypeptide of ~140
kD, close to the predicted size of Gin4p (Fig. 4, lane 1).
This polypeptide was absent in extracts from a gin4-9
strain (Fig. 4, lane 3) and more abundant in extracts from a
strain containing a high-copy GIN4 plasmid (Fig. 4, lane 2), confirming that the purified antibodies are specific for
Gin4p. Second, we introduced a 3HA tag just upstream of
the GIN4 stop codon to create GIN4-3HA (see Materials
and Methods). gin4-9 cells harboring this construct in a
low-copy plasmid expressed Gin4p-3HA at levels similar
to those of the wild-type protein (Fig. 4, compare lanes 1,
4, and 5), and this plasmid fully complemented the morphological defects of a gin4-9 strain (see below, and data
not shown). Third, a bacterial GST gene was introduced
just upstream of the GIN4 stop codon to create GIN4-GST (see Materials and Methods); after integration into
the chromosome, GIN4-GST fully rescued the morphological defects of a gin4-9 strain (see below, and data not
shown). In immunofluorescence experiments, we obtained
essentially identical results using antibodies to Gin4p to
localize Gin4p (Fig. 5 A) or antibodies to HA or GST to
localize Gin4p-3HA (Fig. 5, B and E) or Gin4p-GST (Fig.
6 A), respectively.
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In many unbudded cells, Gin4p was visualized as a single ring in the cell cortex (Fig. 5 E, cells 1, 2, and 6). These
rings appear to correspond to incipient bud sites, as they
were always located at a cell pole with polarized F-actin
(as detected by staining with rhodamine phalloidin; data
not shown), and they corresponded precisely to rings of
septin proteins (Fig. 5, E and F, cells 1, 2, and 6) that had
the appearance (diameter and brightness) of septin rings
marking incipient bud sites rather than of those marking
previous division sites (Kim et al., 1991; Ford and Pringle,
1991). Consistent with this interpretation, when such cells
had two rings of septin proteins, the Gin4p ring always (40 out of 40 cells observed) corresponded to the septin ring
that appeared to be at the incipient bud site (Fig. 5, E and
F, cell 6); unbudded cells with two rings of Gin4p were not observed. Thus, unlike the septins, which typically remain
visible at the division site for some time after cytokinesis
(Kim et al., 1991; Ford and Pringle, 1991), Gin4p seems to
disappear from the neck/division site at about the time of
cytokinesis. Gin4p appears to arrive at the incipient bud
site in late G1, coincident with the arrival of the septins.
Every unbudded cell identified as having a ring of Gin4p-3HA had a corresponding ring of Cdc11p (50 out of 50 cells observed), and nearly every unbudded cell identified
as having a ring of Cdc11p at the incipient bud site had a
corresponding ring of Gin4p-3HA (48 out of 50 cells observed). (The cells with a ring of Cdc11p at the incipient
bud site but no corresponding ring of Gin4p-3HA might
have lost the GIN4-3HA plasmid before immunofluorescence staining.)
Like the septins, Gin4p was localized to the mother-bud
neck throughout the budded phase of the cell cycle (Fig. 5,
A, B, and E). However, in budded cells, the septins are
consistently visualized on both sides of the neck as an apparent double ring (Haarer and Pringle, 1987; Kim et al.,
1991; Ford and Pringle, 1991) (Fig. 5 C; Fig. 5 F, cells 3-5),
whereas Gin4p was visualized as either an apparent single
ring, (Fig. 5 A, cells 1, 2, 4, and 6; Fig. 5 B) or an apparent
double ring (Fig. 5 A, cells 3, 5, and 7; Fig. 5 E, cells 3-5).
The "single rings" of Gin4p were located in the middle or
on the mother side of the neck, within the region occupied
by the septins, and the "double rings" precisely colocalized
with the septins. Careful focusing up and down revealed that Gin4p in double rings was, like the septins, in fact
present as a continuous band throughout the neck region,
at least until very late in the cell cycle; thus, the single ring
versus double ring appearance seems simply to reflect differences in the width of the band of Gin4p along the
mother-bud axis rather than a discrete difference in protein organization. Both "single rings" and "double rings"
of Gin4p were observed at all stages in the cell cycle, but
there was a sharp increase in the frequency of cells with "double rings" at about the time of the G2/M transition
(data not shown).
The overlapping localization of Gin4p and the septins suggested that Gin4p localization may be septin dependent. To test this, a temperature-sensitive cdc12 septin mutant expressing Gin4p-GST and harboring either a control plasmid or a low-copy CDC12 plasmid was grown to exponential phase at 23°C, shifted to 37°C for 45 min, and stained with antibodies to GST or antibodies to Cdc3p. Both Gin4p-GST and Cdc3p remained detectable at the neck in cells containing the CDC12 plasmid (Fig 6, A and B), but both proteins were lost from the neck in cells containing the control plasmid (Fig. 6, C and D). Thus, Gin4p localization to the neck indeed appears to be septin dependent.
To investigate further the determinants of Gin4p localization to the neck, we created plasmids YCpGALGST,
YCpGALGST/GIN4N, and YCpGALGST/GIN4C, which
express GST or fusions of GST to the kinase domain or
the nonkinase region of Gin4p, respectively, under control
of the GAL1/10 promoter. When the proteins were expressed in wild type (strain YEF473) or gin4-9 mutant
(strain M-272) cells and localized by immunofluorescence
using antibodies to GST, GST localized throughout the cytoplasm in a partially punctate pattern (Fig. 6 E), and
GST-Gin4pN localized diffusely throughout the cytoplasm
(Fig. 6 F). In contrast, GST-Gin4pC localized to the neck
(Fig. 6 G), like full-length Gin4p. Thus, the nonkinase region of Gin4p appears to be responsible for localization to the mother-bud neck. Consistent with this hypothesis,
Gin4p kinase activity does not appear to be required for
localization of the full-length protein: when expressed
from a low-copy plasmid (YCp/gin4K48M-3HA) in wild-type or gin4-9 cells or from an integrated single copy
(strain M-692) at room temperature, Gin4pK48M-3HA and
Gin4pK48A-3HA both localized indistinguishably from normal Gin4p or Gin4p-3HA (data not shown).
Effects of Gin4p Overexpression
No obvious effects were seen upon long-term overexpression of GST or of the fusion of GST to the Gin4p kinase
domain (data not shown). However, long-term overexpression of the fusion of GST to the nonkinase region of
Gin4p in wild-type cells, although not lethal, produced
striking morphological abnormalities. Cells frequently had
multiple, elongated buds (Fig. 7, A, B, and E) containing
multiple nuclei that appeared to have segregated efficiently (Fig. 7, C and D). This phenotype was similar to
that of temperature-sensitive septin mutants incubated at
restrictive temperature (Hartwell, 1971; Adams and Pringle, 1984), suggesting that septin localization or assembly
might be defective in these cells. Indeed, septin staining
was usually weak and diffuse compared with that in wild-type cells (Fig. 7 A; compare Fig. 5, C and F), and many
cells lacked detectable septin staining. The localization of
GST-Gin4pC resembled that of the septins (Fig. 7 B). As
expected from the abnormal septin organization (DeMarini et al., 1997) (see Introduction), cells overexpressing
GST-Gin4pC also displayed abnormal patterns of chitin
deposition (Fig 7 E; compare with Fig. 9 G). Similar results
were obtained when GST-Gin4pC was overexpressed in
gin4-9 cells or (in a smaller proportion of the cells) when
wild-type or gin4-9 cells contained a high-copy GIN4 or
gin4K48M plasmid (data not shown). Thus, overexpression
of the nonkinase region of Gin4p, catalytically inactive
Gin4p, or normal Gin4p appears to interfere with septin
localization or assembly.
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Abnormal Morphology and Septin Organization in gin4 Mutants
To characterize further the function of Gin4p, we observed the effects of GIN4 deletion and of mutations that
reduce or eliminate Gin4p kinase activity (see above) on
cell morphology, septin organization, and cytokinesis. In
comparison to wild-type cells (Fig. 8, A and B), most gin4-9 cells grown at 23°C were moderately elongated and
clumped (Fig. 8 C), and many cells had enlarged bud necks (Fig. 8 C, arrows). These abnormalities were more pronounced in gin4-9 cells grown at 37°C (Fig. 8 D); fewer
than 20% of such cells had a wild-type morphology. Cell
clumping at both 30° and 37°C appeared to be due mostly
to a defect in cell separation rather than a defect in cytokinesis, because either sonication or fixation and cell wall
digestion (see Materials and Methods) yielded predominantly single-budded and unbudded cells (data not shown).
Similar abnormalities, but less pronounced, were observed in strains expressing only a kinase-dead GIN4 allele (data
not shown).
Immunofluorescence staining revealed that gin4-9
cells grown at various temperatures were also frequently
abnormal in septin organization. In both diploid and haploid mutant strains, a fraction of cells (ranging from ~61%
at 23°C to ~10% at 37°C) displayed approximately normal
septin staining (Fig. 8 E, cell 3; compare to the wild-type
cells in the inset and in Fig. 5, C and F) (Table V). However, other cells displayed one of several aberrant staining patterns. First, and most strikingly, many cells displayed a
set of five to eight parallel "bars" of septin staining running through the neck along the mother-bud axis (Fig. 8
E, cells 1 and 2; Fig. 8 F) (Table V). Second, many cells
displayed a band of septin staining in the neck whose endpoints were less well defined ("fuzzier") than those in
wild-type cells (Fig. 8 E, cell 4) (Table V). Often (as in Fig.
8 E, cell 4), it appeared that fuzzy bands of septin staining
might actually be poorly resolved sets of bars. (Similarly, the higher ratio of fuzzy bands to bars scored in haploid
cells, relative to diploid cells, may reflect, at least in part, a
lower resolution of structures in the smaller necks of haploid cells.) Both fuzzy and bar septin staining patterns
were observed both in morphologically abnormal cells and
in cells of relatively normal morphology, suggesting that
the abnormalities in septin organization are not simply a
consequence of abnormal cell morphology. Necks with either fuzzy or bar septin staining appeared to contain
amounts of septins comparable to those in wild-type
necks, as judged by the intensity of immunofluorescence
staining; thus, the absence of Gin4p appears to affect the
organization of septins at the neck rather than their ability
to localize to that part of the cell. Third, in some cells, septins were not detectable at the neck (Fig. 8, E, cell 5, and
G, arrow); such cells were rare at 23°C but more common
at higher temperatures (Table V), and their overall morphology typically resembled that of temperature-sensitive septin mutants incubated at restrictive temperature
(Hartwell, 1971; Adams and Pringle, 1984). The absence
of detectable septin staining indeed appeared to correlate
with a defect in cytokinesis: the cytoplasm appeared to be
continuous through necks that were devoid of septin staining, and microtubules could span them (Fig. 8 H); moreover, the two or more nuclei in such cells were typically synchronized in the cell cycle (Fig. 8 I and data not
shown). Occasionally, even necks with septin staining appeared to fail in cytokinesis or cell separation (Fig. 8 E,
cell 6); in these cells, septin staining persisted at the
neck(s) as if septin disassembly had been abnormally delayed.
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Remarkably, however, in most cases, gin4-9 bud necks
with fuzzy or bar septin staining appeared to be competent
to undergo cytokinesis. In gin4-9/gin4-9 diploid cells, at
each temperature examined, the fraction of multibudded
and/or multinucleate cells (indicative of failures of cytokinesis) was much less than the fraction of cells with fuzzy or
bar septin staining (Table V).
To ask if the role of Gin4p in promoting normal septin
organization at the neck depends on the Gin4p kinase activity, we examined cells of a gin4 strain that had been
transformed with plasmids expressing either Gin4p-3HA
or Gin4pK48M-3HA. Although the former plasmid restored
septin organization as effectively as did a plasmid expressing normal Gin4p, the plasmid expressing Gin4pK48M-3HA
only partially restored septin organization (Table V). Similar results were obtained with a strain (M-692) expressing
only Gin4pK48A-3HA. Thus, the Gin4p kinase activity appears to promote, but not to be absolutely essential for,
normal septin organization at the neck.
To investigate further the functional properties of the
abnormally organized septins, we examined the localization and function of Bni4p and Bud4p, two proteins that
localize to the neck in a septin-dependent manner (see Introduction). In wild-type cells, Bni4p is visualized as a
sharp band on the mother-cell side of the neck (DeMarini
et al., 1997) (Fig. 9 A). In contrast, in gin4-9 cells, Bni4p
was typically visualized as a fuzzy band spanning the neck
(Fig. 9 B, arrowheads) (Table V) or as a set of bars similar
to the bars of septin staining (Fig. 9 B, arrows) (Table V);
moreover, in cells grown at 37°C, Bni4p was often undetectable (Table V). At all temperatures tested, aberrant
Bni4p organization was detected more frequently than
was aberrant septin organization (Table V), suggesting
that subtle alterations of septin organization could have
more pronounced (and/or more easily detected) effects on
Bni4p localization or organization. Remarkably, the abnormally organized Bni4p appeared to be functional in assembling components of the chitin synthase III complex:
at 23° or 30°C, most gin4-9 cells displayed either fuzzy or
bar Bni4p staining (Table V), but nearly all cells deposited
chitin primarily at the neck (Fig. 9 H) (Table VI). Strikingly, like Bni4p itself, the chitin was typically present on
both sides of the neck (Fig. 9 H) (Table VI), rather than
being restricted to the mother-cell side of the neck as in
wild-type cells (Fig. 9 G) (Table VI).
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Similar results were obtained with Bud4p. In wild-type
cells, Bud4p localizes to the neck from G2 until the end of
the cell cycle in a tight band that is symmetric on the
mother and bud sides of the neck (Sanders and Herskowitz, 1996). In contrast, in haploid and diploid gin4-9
cells, Bud4p was often visualized in fuzzy or bar patterns
(Fig. 9 C and data not shown) whose frequencies were similar to those observed for Cdc11p (Table V). The abnormally organized Bud4p appeared to be largely functional
in axial bud-site selection: at 30°C, ~36% of the cells displayed Bud4p in a fuzzy or bar pattern (Table V), whereas
only ~6% of the cells budded in nonaxial patterns as
judged by Calcofluor staining of bud scars (see Materials
and Methods). Similarly, at 34°C, ~61% of the cells displayed Bud4p in a fuzzy or bar pattern (Table V), whereas only ~15% of the cells budded in nonaxial patterns.
Interestingly, in cells undergoing polarized morphogenesis (shmooing) in response to mating pheromone, the
septins are also typically visualized either as a fuzzy band
(Kim et al., 1991; Ford and Pringle, 1991) or as a set of
bars parallel to the projection axis (Fig. 9, D and E). This
septin localization corresponds to that of the broad and
somewhat fuzzy band of chitin deposited at the bases of
the shmoo projections (Schekman and Brawley, 1979; Kim
et al., 1991; Konopka et al., 1995) (Fig. 9 I), suggesting that
in shmooing cells, as in vegetative cells (DeMarini et al., 1997), the septins serve as a template for the localization
and/or assembly of Chs3p (Santos and Snyder, 1997) and
the other components of the chitin synthase III complex.
These observations raise the intriguing possibility that the
absence of Gin4p in mutant vegetative cells might result in
an altered organization of the septins that mimics what occurs normally in shmooing cells. Consistent with this hypothesis, Gin4p was not detectable by immunofluorescence in shmooing cells (Fig. 9 F), although the protein
could be detected by Western blotting in such cells (Altman and Kellogg, 1997).
We did not detect any other defects in gin4-9 strains.
Haploid gin4-9 cells mated efficiently with both wild-type and gin4-9 partners, and a diploid gin4-9/gin4-9
strain sporulated with a frequency similar to that of an
otherwise isogenic wild-type strain and produced ascospores of normal viability.
Apparent Lack of Redundancy between Gin4p and Ycl024Wp
To investigate the function of the Gin4p homologue
Ycl024Wp, its ORF was precisely replaced with HIS3 (see
Materials and Methods). Haploid and homozygous diploid
ycl024W-2 strains were viable and displayed no readily
detectable defects in mating, cell morphology, chitin deposition, bud-site selection, sporulation, spore germination, growth on YPD plates at temperatures ranging from 18° to
39°C, or growth on plates containing 1 M sorbitol, 0.4 M
KCl, or 0.9 M KCl (data not shown). Moreover, gin4-9
ycl024W-2 haploid and homozygous diploid double-mutant strains were indistinguishable from the gin4-9 single-
mutant strains; they displayed neither enhanced nor novel
phenotypes. Thus, somewhat surprisingly, Ycl024Wp does
not appear to be redundant, or to share an overlapping
function, with Gin4p.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Identification of Septin-interacting Proteins by a Synthetic-lethal Screen
A screen for mutations lethal in combination with the
cdc12-5 septin mutation has identified several genes whose
products interact with the septins (DeMarini et al., 1997;
this study).2 In the work reported here, we found that loss
of function mutations in either of two protein kinase
genes, GIN4 and MPK1/SLT2, are synthetically lethal
with cdc12 mutations. As discussed below, abundant evidence demonstrates an intimate interaction between Gin4p and the septins. In contrast, it remains unclear how
directly Mpk1p and the septins interact. mpk1 cdc12-6
double mutants arrest growth with cells at various stages
in the cell cycle and do not resemble a septin loss of function mutant in morphology; thus, it does not appear that
the absence of Mpk1p simply compromises septin function. As both Mpk1p (Cid et al., 1995; Levin and Errede, 1995; Banuett, 1998) and the septins (Hartwell, 1971; Adams and Pringle, 1984; Longtine et al., 1996; DeMarini et
al., 1997) are involved in the control of cell-surface growth,
the synthetic lethality may reflect a synergistic effect on
the maintenance of cell integrity.
Interaction of Gin4p and the Septins
Many lines of evidence show that Gin4p interacts intimately with the septins and plays a positive role in septin
function. First, these proteins colocalize through most of
the cell cycle. Both Gin4p and the septins assemble into a
ring at the presumptive bud site ~15 min before bud
emergence (this study; Ford and Pringle, 1991; Kim et al.,
1991), and both do so in an actin-independent manner
(Ayscough et al., 1997). The proteins then remain concentrated in a band at the mother-bud neck throughout the
period of bud growth (this study; Okuzaki et al., 1997). Interestingly, the band of Gin4p often occupies only a part of
the region defined by the band of septins, and, unlike the
septins (Ford and Pringle, 1991; Kim et al., 1991) (Fig. 5 F,
cell 6), Gin4p disappears from the neck at about the time
of cell division. These observations suggest that Gin4p localization might depend on the septins but not vice versa.
Consistent with this hypothesis, Gin4p localization is lost
when a septin mutant is shifted to restrictive temperature, whereas the septins still generally localize to the neck (albeit in abnormal patterns; see below) in the absence of
Gin4p. Second, interaction between Gin4p and the septins
has been observed both in the two-hybrid system (Table
IV) and by affinity chromatography (Carroll et al., 1998).
Third, deletion of GIN4 in a wild-type background, although not lethal, results both in a dramatically altered reorganization of the septins (as discussed more fully below)
and in a mutant cell morphology that suggests that septin function is compromised. Fourth, gin4 and cdc12 mutations
are synthetically lethal, and the morphology of the double
mutants suggests that septin function is lost in the absence
of Gin4p. Fifth, mild overexpression of GIN4 can rescue
both the viability and the morphology of a cdc12 temperature-sensitive mutant at intermediate temperatures. Finally, more extreme overexpression of full-length Gin4p or
(especially) of a COOH-terminal Gin4p fragment causes
disorganization of the septins and an abnormal cell morphology that resembles that of septin mutants.
The protein kinase activity of Gin4p appears to be important for its positive role in septin function, as kinase-dead mutants of Gin4p can rescue neither the cdc12 single
mutant at intermediate temperatures nor the synthetic lethality of the gin4 cdc12 double mutant, and strains expressing only a kinase-dead allele have many cells with
conspicuously abnormal septin organization (Table V).
Moreover, the two-hybrid interaction observed was between the COOH-terminal region of Cdc3p and the kinase
domain of Gin4p; this interaction appeared to depend on
the presence of an intact kinase domain, as a construct
lacking only the NH2-terminal 17 amino acids of Gin4p
failed to interact (Table IV; note the caveat that negative
results in two-hybrid assays can have a variety of causes).
As two-hybrid interactions have been detected between other protein kinases and their substrates (Yang et al.,
1992; Staudinger et al., 1995; Cook et al., 1996) and Cdc3p
appears to be phosphorylated in vivo (Healy, A.M., and
J.R. Pringle, unpublished results; Reed, S.I., personal comm
109 and +517,
respectively, relative to the
beginning of the 3426-nucleotide GIN4 ORF. Plasmids
p
