HTL1 encodes a novel factor that interacts with the RSC chromatin remodeling complex in Saccharomyces cerevisiae.

Abstract

RSC is an essential chromatin remodeling complex in Saccharomyces cerevisiae that performs central roles in transcriptional regulation and cell cycle progression. Here we identify Htl1 as a novel factor that associates with the RSC complex both physically and functionally. We isolated HTL1 through a genetic screen for mutants that displayed additive growth defects with a conditional mutation in the protein kinase C gene (PKC1), which has been suggested through genetic connections to interact functionally with RSC. Several lines of evidence connect HTL1 to RSC function. First, an htl1Delta mutant displayed temperature-sensitive growth and a G(2)/M cell cycle arrest at restrictive temperatures, a phenotype similar to that of strains with conditional mutations in essential RSC components. Second, we isolated RSC3, which encodes a component of the RSC complex, as a dosage suppressor of the htl1Delta growth arrest. Third, an htl1Delta mutant displayed additive growth defects with conditional rsc3 alleles. Fourth, overexpression of HTL1 suppressed the growth defect of a strain with a conditional mutation in another RSC component, RSC8. Finally, we demonstrate that Htl1 is a nuclear protein that can associate in vivo with a fraction of the RSC complex. We propose that an RSC-Htl1 complex acts coordinately with protein kinase C to regulate the G(2)/M transition.

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MOLECULAR AND CELLULAR BIOLOGY, Dec. 2002, p. 8165–8174
0270-7306/02/$04.00?0DOI: 10.1128/MCB.22.23.8165–8174.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 23
HTL1 Encodes a Novel Factor That Interacts with the RSC Chromatin
Remodeling Complex in Saccharomyces cerevisiae
Martin J. Romeo,1Melinda L. Angus-Hill,2Andrew K. Sobering,1† Yoshiaki Kamada,1‡
Bradley R. Cairns,2and David E. Levin1*
Department of Biochemistry & Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University,
Baltimore, Maryland 21205,1and Howard Hughes Medical Institute, Department of Oncological Sciences,
University of Utah School of Medicine, Salt Lake City, Utah 841122
Received 20 June 2002/Returned for modification 5 August 2002/Accepted 26 August 2002
RSC is an essential chromatin remodeling complex in Saccharomyces cerevisiae that performs central roles
in transcriptional regulation and cell cycle progression. Here we identify Htl1 as a novel factor that associates
with the RSC complex both physically and functionally. We isolated HTL1 through a genetic screen for mutants
that displayed additive growth defects with a conditional mutation in the protein kinase C gene (PKC1), which
has been suggested through genetic connections to interact functionally with RSC. Several lines of evidence
connect HTL1 to RSC function. First, an htl1? mutant displayed temperature-sensitive growth and a G2/M cell
cycle arrest at restrictive temperatures, a phenotype similar to that of strains with conditional mutations in
essential RSC components. Second, we isolated RSC3, which encodes a component of the RSC complex, as a
dosage suppressor of the htl1? growth arrest. Third, an htl1? mutant displayed additive growth defects with
conditional rsc3 alleles. Fourth, overexpression of HTL1 suppressed the growth defect of a strain with a
conditional mutation in another RSC component, RSC8. Finally, we demonstrate that Htl1 is a nuclear protein
that can associate in vivo with a fraction of the RSC complex. We propose that an RSC-Htl1 complex acts
coordinately with protein kinase C to regulate the G2/M transition.
Changes in chromatin structure play an important and dy-
namic role in gene regulation (19, 23). Chromatin remodelers
are multiprotein complexes comprising an ATPase “engine”
required for nucleosome repositioning (4, 24, 48) and various
additional components thought to be important for the regu-
lation and targeting of such complexes to genomic loci (38).
Chromatin remodeling complexes are thought to activate or
repress gene expression by exposing or concealing regulatory
sequences. Two such complexes in Saccharomyces cerevisiae
include the SWI/SNF complex and the RSC complex (named
RSC for remodels the structure of chromatin). RSC is an
abundant complex of at least 16 subunits, which was purified
on the basis of homology of several of its subunits to those of
the SWI/SNF complex (5). Of the two, only RSC is essential
for viability, serving an important function in cell cycle pro-
gression from G2to M (1, 5, 7, 45).
The RSC complex exists in distinct forms, containing either
Rsc1 or Rsc2, and with or without Rsc3 and Rsc30 (1, 5, 6).
Moreover, the RSC subunit Sfh1 is phosphorylated during the
G1phase of the cell cycle (7), suggesting that chromatin re-
modeling by RSC is regulated at multiple levels. Additionally,
rsc mutations affect expression of genes involved in cell wall
biogenesis, ribosome biogenesis, the nitrogen discrimination
pathway (NDP), carbon source utilization, and the TOR path-
way. Recently, whole-genome occupancy studies have revealed
the occupancy of RSC at hundreds of yeast genes, including
those involved in NDP, carbon source utilization, the TOR
pathway, histone genes, and tRNA genes (9, 34).
Genetic observations have revealed a functional link be-
tween RSC components and the cell wall integrity signaling
pathway controlled by the Rho1 GTPase and Pkc1 (1, 7a, 14).
This pathway monitors and regulates cell wall biogenesis dur-
ing vegetative growth and in response to pheromone-induced
morphogenesis (31). The master switch for cell wall signaling is
Rho1, which is activated by several members of a family of cell
surface sensors (12, 17, 22, 36, 39, 46).
Active Rho-GTP binds to and activates protein kinase C (21,
35), which is encoded by PKC1 (32). Loss of PKC1 function, or
any of the components of the mitogen-activated protein
(MAP) kinase cascade under its control (31), results in a cell
lysis defect that is attributable to a deficiency in cell wall con-
struction (29, 30, 37). The MAP kinase cascade is a linear
pathway comprised of a MEKK (Bck1 [8, 27]), a pair of re-
dundant MEKs (Mkk1/2 [15]), and a MAP kinase (Mpk1/Slt2
[26, 33]). One consequence of signaling through the MAP
kinase cascade is the activation of the Rlm1 transcription fac-
tor (10, 47). Signaling through Rlm1 regulates the expression
of at least 25 genes, most of which have been implicated in cell
wall biogenesis (18).
Because the growth defect of a pkc1 null mutant is more
severe than that of any of the pathway components that func-
tion downstream of this protein kinase, we have proposed that
Pkc1 regulates a bifurcated pathway (30). To elucidate the
nature of the second pathway branch and to identify novel
targets of Pkc1, we conducted a screen for mutations that
* Corresponding author. Mailing address: Department of Biochem-
istry & Molecular Biology, The Johns Hopkins University, Bloomberg
School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205.
Phone: (410) 955-9825. Fax: (410) 955-2926. E-mail: dlevin1@jhem
.jhmi.edu.
† Present address: Institut de Genetique et Microbiologie, Univer-
site ´ Paris-Sud, 91405 Orsay Cedex, France.
‡ Present address: Department of Cell Biology, National Institute
for Basic Biology, Okazaki 444-8585, Japan.
8165

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displayed additive growth defects with a pkc1tsmutation. Here
we report the isolation of HTL1, a gene previously reported to
be required for genomic stability and for growth at elevated
temperatures (25). We demonstrate that HTL1 is important
for cell cycle progression. Loss of function of HTL1 results in
a G2/M arrest at restrictive temperatures that is similar to that
observed in strains with conditional mutations of essential RSC
subunits. Finally, we show that Htl1 interacts with the RSC
complex both physically and functionally.
MATERIALS AND METHODS
Strains, growth conditions, and transformations. The S. cerevisiae strains used
in this study are listed in Table 1. Yeast cultures were grown in YEPD (1% Bacto
yeast extract, 2% Bacto Peptone, 2% glucose) with or without 10% sorbitol.
Synthetic minimal (SD) medium (40) supplemented with the appropriate nutri-
ents was used to select for plasmid maintenance and gene replacement. Synthetic
complete (SC) medium with or without 5-fluoroorotic acid (5-FOA) (0.1% [3])
was used to assess the viability of rsc3 htl1? mutants. Yeast cells were trans-
formed by the lithium acetate method (16). Escherichia coli DH5? was used to
propagate all plasmids. E. coli cells were cultured in Luria broth medium (1%
Bacto Tryptone, 0.5% Bacto yeast extract, 1% NaCl) and transformed by stan-
dard methods.
Synthetic lethal screen and isolation of HTL1. S. cerevisiae strain DL1248 was
grown in SD medium to an A600of 0.5, washed, and resuspended in 0.9% KCl to
a density of 3 ? 107cells/ml. The cells were then irradiated for 60 s with a
254-nm-wavelength UV lamp (0.5 J m?2s?1), which resulted in an approxi-
mately 25% loss in plating efficiency. The irradiated cells were harvested, resus-
pended in YEPD, and grown overnight in the dark at 23°C. They were then
plated onto YEPD (approximately 150 colonies/plate) and allowed to grow at
23°C for 3 to 4 days. Colonies that appeared entirely red with no white sectors
were picked, patched onto YEPD, and grown at 37°C for 2 to 3 days. Five
temperature-sensitive isolates were transformed with a centromeric library (in
pRS314), plated on SD medium lacking tryptophan, and incubated at 23°C
overnight. Plates were then shifted to 37°C for two additional days to select for
complementing clones. Plasmids were rescued from colonies arising at 37°C. Of
the five original temperature-sensitive synthetic lethal isolates, two (DL2822 and
DL2823) were complemented by single plasmids with overlapping sequences
from the right arm of chromosome III. The plasmid (designated A3) rescued
from DL2822 possessed a 6.6-kb insert that contained five complete open read-
ing frames (ORFs) (PET18, MAK31, HTL1, HSP30, and YCR022C), 1,092 bp of
the 3? end of MAK32, and 924 bp of the 3? end of YCR023C. The plasmid rescued
from DL2823 (designated A4) possessed an insert of approximately 5.4 kb that
contained five complete ORFs (MAK32, PET18, MAK31, HTL1, and HSP30), a
partial Ty element, and 296 bp of the 3? end of YCR022C. A 527-bp EcoRV-
HindIII fragment containing only HTL1/YCR020W-B was the smallest segment
that allowed sectoring of strains DL2822 and DL2823 at 23°C and growth at
37°C. This fragment was subcloned into a 2?m plasmid, YEp352 (13), and a
centromeric plasmid, pRS316 (42).
Southern blotting for HTL1. The 666-bp HindIII fragment containing HTL1
was32P labeled using the Multiprime random priming kit (Invitrogen Life Tech-
nologies). This labeled fragment was used to probe HindIII-digested genomic
DNA (5 ?g) from strains DL1248 or DL2822 separated on an agarose gel and
transferred to a nitrocellulose membrane.
Genomic deletion of HTL1. To disrupt the genomic copy of HTL1, 795 bp of
sequence 5? to the HTL1 start codon and 1,030 bp of sequence 3? of the HTL1
stop codon were amplified in separate PCRs from genomic DNA of strain 1783.
The 5? fragment was amplified with primers that placed a NotI site at the end
adjacent to the HTL1 coding sequence and a BamHI site at the opposite end.
The 3? fragment was amplified with primers that placed a KpnI site adjacent to
the HTL1 coding sequence and a BamHI site at the opposite end. These frag-
ments were ligated in a three-molecule reaction to the NotI and KpnI sites of the
integrative plasmid pRS304 (42) to create a unique BamHI site between the
fragments. The resulting plasmid, pRS304[htl1?::TRP1], was linearized with
BamHI and used to transform the yeast strain to tryptophan prototrophy. De-
letion of HTL1 in Trp?transformants was confirmed by PCR. All primers were
obtained from Invitrogen Life Technologies.
Isolation of RSC3 as a dosage suppressor of the htl1? growth defect. A diploid
yeast strain (DL2754 [htl1?/htl1?]) was used to avoid isolation of recessive
suppressor mutations. This strain was cultured at 23°C in 50 ml of YEPD to an
A600of 0.8. Cells were transformed with a 2?m URA3-marked library (in
pRS202; gift of F. Spencer) and plated on SD medium lacking uracil. Plates were
incubated at 23°C for 1 day and shifted to 34°C for 2 to 3 days to select directly
for suppressors. The smallest insert identified that was capable of suppressing the
growth defect was a 4.3-kb fragment with one complete ORF (RSC3), 264 bp of
the 3? end of CPR5, and 551 bp of the 3? end of GPI11. A 3.4-kb PvuII/MscI
fragment containing RSC3 was subcloned into the SmaI site of the 2?m vector
TABLE 1. S. cerevisiae strains used in this study
Strain Relevant genotypeSource or reference
1783
1788
DL376
DL1248
DL2312
DL2315
DL2745
DL2749
DL2751
DL2754
DL2822
DL2823
MCY3890
YBC628
YBC840
YBC842
YBC843
YBC906
YBC928
YBC1334
YBC1336
YBC1338
YBC1340
MATaa
MATa/MAT?a
MATa pkc1?::LEU2a
MAT? stt1-1 pRS315[PKC1 ADE3]b
MATa bck1?::HIS4a
MATa/MAT? bck1?::HIS4/BCK1a
MATa bck1?::HIS4 htl1?::TRP1a
MATa htl1?::TRP1a
MAT? htl1?::TRP1a
MATa/MAT? htl1?::TRP1/htl1?::TRP1a
MAT? “pet18” isolate of DL1248#1b
MAT? “pet18” isolate of DL1248#2b
MATa swh3-ts16 (rsc8ts)c
MATa rsc3?::HIS3 pRS316[RSC3]d
MATa rsc3?::HIS3 pRS315[rsc3-1]d
MATa rsc3?::HIS3 pRS315[rsc3-2]d
MATa rsc3?::HIS3 pRS315[RSC3]d
MATa rsc3?::HIS3 pRS315[rsc3-3]d
MATa his3?1 leu2?0 lys2?0 ura3?0 trp1?63 pep4?::Kanmx
MATa rsc3?::HIS3 htl1?::TRP1 pRS316[HTL1] pRS315[RSC3]d
MATa rsc3?::HIS3 htl1?::TRP1 pRS316[HTL1] pRS315[rsc3-1]d
MATa rsc3?::HIS3 htl1?::TRP1 pRS316[HTL1] pRS315[rsc3-2]d
MATa rsc3?::HIS3 htl1?::TRP1 pRS316[HTL1] pRS315[rsc3-3]d
I. Herskowitz
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29
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aEG123 background (leu2-3,112 trp1-1 ura3-52 his4 can1).
bade2 ade3 ura3 leu2 trp1 his3.
cW303-1A background (trp1-1 leu2-3,112 his3-11,15 ura3-1 ade2-1 can1-100).
dhis3?200 leu2?1 lys2-128? trp1?63 ura3-52.
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pRS426. This clone was capable of suppressing the temperature sensitivity of
strain DL2754.
FACS analysis. Cultures of strains 1788 and DL2754, grown in YEPD plus
10% sorbitol at 23°C, were diluted to A600of 0.2 with an equal volume of YEPD
plus 10% sorbitol prewarmed to 54°C and cultured at 38°C for either 5 or 10 h
(DL2754 loses viability upon longer maintenance at restrictive temperatures).
The wild-type culture (strain 1788) was periodically diluted with prewarmed
medium to maintain the cells in the logarithmic growth phase. Aliquots of 1 ml
were drawn after 5 and 10 h at restrictive temperatures and prepared for fluo-
rescence-activated cell sorting (FACS) analysis by a wash with phosphate-buff-
ered saline (PBS) followed by overnight fixation in 70% ethanol at 4°C. Cells
were washed with PBS, treated extensively with RNase A (1 mg/ml for 5 h at
37°C), washed again, and stained with 50 ?g of propidium iodide per ml. Cells
were sonicated briefly and diluted to a final concentration of 106cells/ml for
FACS analysis, which was performed with a Becton Dickinson FACSCalibur
cytometer at the Johns Hopkins Flow Cytometry Core Facility. Another 1-ml
aliquot of each culture was drawn at each time point for microscopy, washed with
PBS, and resuspended in VectaShield with 4?,6?-diamidino-2-phenylindole
(DAPI) (1 ?g/ml) for microscopic analysis.
Construction of HA- and FLAG-tagged HTL1. To create HTL1-3xHA, HTL1
was PCR amplified from an HTL1 plasmid using primers that introduced a SacI
site 794 bp 5? to the HTL1 translational start site and omitted the stop codon
from the HTL1 coding sequence. This fragment was introduced into
YEp352[3xHA] (39), creating an in-frame fusion at the 3? end of HTL1, with a
glycyl codon between the HTL1 and hemagglutinin (HA)-coding sequences. This
epitope-tagged form of Htl1 (Htl13xHA) was fully functional, as judged by its
ability to complement the htl1? growth defect when expressed from a centro-
meric plasmid. This construction was used for indirect immunofluorescence
microscopy with a Zeiss Axioskop microscope, as described previously (20). To
createFlagHTL1, HTL1 was PCR amplified with a forward primer designed to
contain a BamHI site immediately upstream of the Flag coding sequence (8
amino acids preceded by an ATG) followed by 24 bp of HTL1 sequence (omit-
ting the endogenous start codon). The reverse primer was designed to place an
EcoRI site downstream of the HTL1 stop codon. The PCR product was intro-
duced into pRS426 containing the methionine-repressible MET25 promoter and
the CYC1 transcriptional terminator (p426[MET25]) to yield pMET-Flag-HTL1.
This plasmid complemented the htl1? growth defect even when expression of
FlagHtl1 was repressed with methionine.
Association of Flag-tagged Htl1 with RSC complex. Yeast strain YBC928 was
transformed to uracil prototrophy with either pMET-Flag-HTL1 or empty vector.
Transformants were cultured at 28°C in YEPD; extracts were prepared as de-
scribed previously (6). For immunoprecipitation ofFlagHtl1, anti-Flag M2 affinity
gel or control beads (Sigma) were incubated for 2 h with 300 ?g of protein extract
in immunoprecipitation (IP) buffer A (50 mM Tris-Cl [pH 7.5], 10% glycerol, 100
mM NaCl, 2 mM EDTA, ?-mercaptoethanol). Precipitates were recovered and
washed in IP buffer A containing 250 mM NaCl and 0.05% Tween 20 and eluted
with 0.2 mg of Flag peptide (Sigma) per ml in IP buffer A. Proteins from crude
extracts, Flag-Htl1-depleted supernatant fractions, and eluates were separated
on a gradient sodium dodecyl sulfate (SDS)–10 to 20% (acrylamide) polyacryl-
amide gel (Bio-Rad), and transferred to polyvinylidene difluoride membranes
for immunoblot detection ofFlagHtl1 with anti-Flag antibodies (Sigma) and of
Sth1 with anti-Sth1 antibodies (1). The extracts described above were also used
for immunoprecipitation of Sth1. Anti-Sth1 antibodies were conjugated to pro-
tein A-Sepharose beads (Sigma) at a concentration of 0.5 mg/ml in IP buffer A;
the beads and antibodies h were then incubated for 2 h with 300 ?g of protein
extract and treated as described above, except that immunoprecipitates were
eluted with SDS sample buffer.
RESULTS
Mutation of HTL1is synthetically lethal with pkc1. To iden-
tify genes whose loss of function results in an additive growth
defect with pkc1, we constructed a strain (DL1248) that har-
bors a genomic pkc1tsallele (stt1-1 [49]), ade2 and ade3 muta-
tions, and a plasmid bearing wild-type copies of both PKC1 and
ADE3. This strain can survive loss of the PKC1-ADE3 plasmid
at 23°C (but not at 37°C), yielding white sectors within red
colonies. Mutants of this strain that cannot survive loss of the
plasmid-borne PKC1, and therefore fail to form sectors, were
isolated after UV mutagenesis. Of 150 nonsectoring colonies
isolated, five were identified as temperature sensitive for
growth at 37°C, indicating the presence of a new temperature-
sensitive mutation. This secondary phenotype facilitated isola-
tion of the affected gene. These mutants were transformed
with a centromeric library of genomic yeast DNA (27) to iso-
late genes capable of complementing their temperature-sensi-
tive growth defects. Of these mutants, one was complemented
by MPK1, one was complemented by PKC1, and one was com-
plemented by a previously uncharacterized nonannotated
ORF (designated RIN1 [A. K. Sobering, M. J. Romeo, H. A.
Vay, and D. E. Levin, submitted for publication]). The remain-
ing two mutants (DL2822 and DL2823) were both rescued by
plasmids (designated A3 and A4, respectively) containing
overlapping fragments from the right arm of chromosome 3,
with PET18, MAK31, HTL1, and HSP30 in common. Deletion
analysis of these plasmids revealed that the smallest segment
allowing sectoring of strains DL2822 and DL2823 at 23°C
and growth at 37°C was a 527-bp EcoRV-HindIII fragment
containing only HTL1/YCR020W-B (named HTL for high
temperature lethal [25]) (Fig. 1A). The HTL1 gene encodes
a recently identified 78-amino-acid protein with no known ho-
mologs.
To determine if strains DL2822 and DL2823 bear mutations
in HTL1, we attempted to PCR amplify from these strains the
genomic regions containing HTL1. However, we were not able
to isolate PCR products from either mutant even when using
primers up to 3 kb flanking either side of HTL1, suggesting
that HTL1 had been deleted from their genomes. Previously
described PET18 mutants (28) resulted from spontaneous de-
letions of greater than 10 kb from the right arm of chromo-
some 3 (43). Such mutants are temperature sensitive due to
loss of HTL1 (25) and unable to grow on nonfermentable
carbon sources. The latter phenotype is known to result from
loss of function of another, as yet unidentified gene within the
deleted region (25). We found that like the previously de-
scribed mutants, strains DL2822 and DL2823 were unable to
grow on rich medium containing glycerol as the sole carbon
source (Fig. 1A). This defect was not complemented by either
plasmid A3 (bearing PET18, MAK31, HTL1, HSP30, and
YCR022C), or plasmid A4 (bearing MAK32, PET18, MAK31,
HTL1, and HSP30), indicating that this strain had suffered a
genomic deletion that extends beyond the region from MAK32-
YCR022C. A Southern blot of genomic DNA verified the ab-
sence of HTL1 in DL2822 (Fig. 1B). Taken together, these
results suggest that loss of HTL1 function is synthetically lethal
with stt1-1.
To examine the behavior of a null mutation in HTL1 alone,
we constructed a genomic deletion of HTL1 in wild-type strain
1788. Heterozygous htl1?::TRP1 diploids were induced to
sporulate, and haploid segregants were dissected. Tryptophan
prototrophy cosegregated with temperature sensitivity for
growth at or above 34°C, consistent with the previous report by
Lanzuolo et al. (25). As anticipated, the htl1? mutants were
capable of growth on glycerol-containing medium (Fig. 1A).
We next tested for additive growth defects caused by com-
bining null mutations in HTL1 and either PKC1 or BCK1.
Doubly heterozygous diploids were generated by crossing an
htl1?::TRP1 strain (DL2751) with a pkc1?::LEU2 strain
(DL376). A total of 56 tetrads were dissected and allowed to
germinate at 23°C on medium containing 10% sorbitol for
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osmotic support. Although both single mutants and wild-type
segregants were recovered at the expected frequencies, no
viable Leu?Trp?spores were recovered, indicating that the
combined loss of PKC1 and HTL1 functions is lethal. HTL1
was also deleted in a heterozygous bck1?/BCK1 diploid
(DL2315). The resultant transformants were induced to sporu-
late, and haploid segregants germinated at 23°C. Although the
bck1? htl1? segregants were able to grow on YEPD at 23°C,
they failed to grow at or above 30°C (Fig. 2). This restrictive
temperature was 4°C lower than that of the htl1? mutant,
which grew poorly at 30°C. This result indicates only mild
additivity of the htl1? and bck1? growth defects. Additionally,
the double mutant displayed a cell lysis defect, as judged by the
presence of nonrefractile ghosts (not shown) that was similar
FIG. 1. A deletion through HTL1 confers an additive growth defect with pkc1ts. (A) The mutation in strain DL2822 results in separable growth
defects at 34°C and on medium with glycerol as the sole carbon source. Strain DL2822, transformed with centromeric plasmids bearing HTL1
alone, plasmid A3 (PET18, MAK31, HTL1, HSP30, and YCR022C), plasmid A4 (MAK32, PET18, MAK31, HTL1, and HSP30), or vector (pRS314),
was streaked onto YEP plus glucose or YEP plus glycerol and incubated at the indicated temperatures for 3 days. The parental strain, DL1248,
and an htl1? strain (DL2751) are included. (B) HTL1 has been lost from the genome of DL2822. Genomic DNA (5 ?g) from strain DL2822 and
parental strain DL1248 was digested with HindIII, separated on an agarose gel, and subjected to Southern blot analysis using the 666-bp HindIII
fragment bearing HTL1 as a probe (top blot). The bottom blot shows an ethidium bromide (EtBr)-stained ribosomal repeat fragment from the
same gel as a quantitation control.
FIG. 2. Null mutations in BCK1 and HTL1 result in mildly additive growth defects. Wild-type (WT) (1783), bck1? (DL2312), htl1? (DL2749),
and bck1? htl1? (DL2745) yeast strains were streaked onto YEPD, plated, and incubated at the indicated temperatures for 3 days.
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to that of bck1? mutants at restrictive temperatures. However,
we were not able to detect a cell wall deficiency in htl1?
mutants using tests for hypersensitivity to the cell wall lytic
enzyme zymolyase or the cell wall antagonist caffeine (data not
shown).
A null HTL1 mutant undergoes a cell cycle-specific growth
arrest at nonpermissive temperatures. Microscopic examina-
tion of htl1? cells at restrictive temperatures revealed that this
mutant arrested growth with a nearly uniform morphology. For
all mating types, 80 to 90% of cells arrested with single, me-
dium- to large-sized buds, single nuclei, and no evidence of cell
lysis (Fig. 3A). To determine whether htl1? cells arrest in the
cell division cycle before or after DNA replication, we assessed
the DNA content of this mutant cultivated at either low or high
temperatures. Log-phase cultures of diploid wild-type (1788)
or htl1? cells (DL2754) were shifted from 23 to 38°C for 5 or
10 h to measure DNA content by FACS analysis. The htl1?
mutant displayed an enriched population of 4N cells at low
temperatures compared to the wild-type population, suggest-
ing a delay at the G2/M boundary (Fig. 3B). Moreover, at
restrictive temperatures, 90% of the htl1? cells accumulated
4N nuclei, indicating a postreplicative arrest at G2/M. Similar
results were obtained with haploid htl1? cells (data not
shown).
RSC3 is a dosage suppressor of htl1?. To gain an under-
standing of the htl1? growth defect, we isolated multicopy
suppressors of an htl1?/htl1? mutant (DL2754). Suppressors
were selected directly at 34°C on SD plates lacking uracil after
transformation with a 2?m URA3-marked genomic library. Of
?80,000 transformants selected, 8 colonies arose at the restric-
tive temperature. Among the plasmids rescued from these
colonies, four contained wild-type HTL1, and four possessed
overlapping DNA fragments from the right arm of chromo-
some IV. The only complete gene common to all four plasmids
was RSC3 (named RSC for remodels the structure of chroma-
tin) (data not shown). Deletion analysis revealed that RSC3
alone, when expressed from a 2?m plasmid, was sufficient to
allow an htl1?/htl1? mutant to grow at 34°C (Fig. 4A). The
RSC3 gene encodes an essential component of the RSC com-
plex, which alters gene expression by remodeling of chromatin
(1). Rsc3 is highly similar to well-characterized fungal DNA-
binding proteins (such as Gal4), as it contains an essential
binuclear zinc cluster followed by a leucine zipper. Some genes
involved in the maintenance of cell wall integrity are among
those regulated by RSC (1). Interestingly, conditional muta-
tions in RSC3 and other essential genes encoding RSC mem-
bers arrest growth at the G2/M boundary (1, 7, 9, 45), with a
terminal morphology that is very similar to that observed for
htl1? cells. However, overexpression of HTL1 failed to sup-
FIG. 3. Deletion of HTL1 results in a cell cycle-specific growth
arrest at restrictive temperatures. (A) Phase-contrast microscopy and
fluorescence microscopy (DAPI staining) of log-phase cultures of
DL2754 (htl1?/htl1?) growing at 23°C or arrested for 10 h at 38°C. The
fractions of cells that appear to have single buds (S), no buds (unbud-
ded [U]), or multiple buds (MB) were determined from counts of at
least 100 cells. (B) Flow cytometric analysis of cultures of either strain
1788 (wild type [WT]) or DL2754 incubated at 23°C or shifted to 38°C
for the indicated times.
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press the growth defects of any of the three conditional rsc3
alleles.
Rsc30 is an RSC subunit that is almost identical to Rsc3 in
the binuclear zinc cluster region. Several lines of evidence
suggest that Rsc3/30 operate as a heterodimer within the RSC
complex but perform different functions (1). In contrast to
RSC3, overexpression of RSC30 from a 2?m plasmid did not
suppress the growth defect of htl1?/htl1? mutants (data not
shown), consistent with the different roles these components
serve in RSC function.
Deletion of HTL1 results in additive growth defects with rsc3
mutations. Because RSC3 is an essential gene, we tested a set
of conditional rsc3 mutations for additive growth defects with
htl1?. The htl1? mutation was introduced into strains bearing
one of three temperature-sensitive alleles of RSC3 (rsc3-1, -2,
and -3 [1]) and a centromeric URA3-based plasmid with HTL1.
These strains were tested for the ability to survive loss of the
plasmid-borne HTL1 at 23°C by plating on 5-FOA-containing
medium to evict the plasmid. Figure 4B shows that both the
rsc3-2 htl1? and rsc3-3 htl1? mutants were unable to grow on
5-FOA-containing medium, indicating that loss of HTL1 is
lethal in these mutants. Together with the observation that
RSC3 is a dosage suppressor of htl1?, these results suggest that
HTL1 and RSC3 function within the same pathway.
HTL1 is a dosage suppressor of an rsc8tsmutant. The RSC8
gene encodes a component of the RSC complex, and homologs
of Rsc8 are present in all SWI/SNF family remodeling com-
plexes (5, 44). Though the precise role of Rsc8 is not known,
studies on its human homolog, BAF155/170, suggest that this
subunit interacts with the ATPase subunit Brg1 and also with
gene-specific transcriptional activators such as EKLF (2).
Treich et al. (44) reported previously that a DNA fragment
bearing the MAK31 and HTL1 loci could suppress the growth
defect of an rsc8tsmutant (swh3-ts16) when maintained at high
copy number. To test whether this suppression was the result
of HTL1 overexpression, a 2?m plasmid bearing only HTL1
was introduced to the swh3-ts16 mutant (MCY3890 [44]). This
clone was able to suppress the growth defect of swh3-ts16 (Fig.
4C), demonstrating that HTL1, not MAK31, is the multicopy
suppressor of swh3-ts16 obtained by Treich et al. (44), and
further supporting the notion that Htl1 functions in the same
pathway as the RSC complex.
Htl13xHAresides in the nucleus. The RSC complex resides in
the nucleus (9, 45). As an initial step toward establishing a
physical connection between Htl1 and RSC, we determined the
intracellular location of Htl1. Htl1 was fused at its C terminus
to a triple-HA epitope (3xHA). Htl13xHAexpressed from a
2?m plasmid colocalized with DAPI, indicating nuclear local-
FIG. 4. Genetic connections between HTL1 and RSC. (A) Overexpression of RSC3 suppresses the growth defect of an htl1? mutant. Strain
DL2754 (htl1?/htl1?) harboring 2?m plasmid pRS426 [RSC3], centromeric plasmid pRS316 [HTL1], or vector (pRS426) were streaked onto
YEPD and incubated at 34°C for 3 days. (B) Deletion of HTL1 results in additive growth defects with conditional rsc3 mutations. Yeast strains
(from top to bottom, YBC628, YBC843, YBC1334, YBC840, YBC1336, YBC842, YBC1338, YBC906, and YBC1340) were grown in selective
medium at room temperature overnight. The saturated cultures and a series of 10-fold dilutions were spotted onto SC plates or SC plates
containing 5-FOA and incubated at 28°C for 3 days. (C) Overexpression of HTL1 suppresses the growth defect of an rsc8tsmutant. Yeast strain
MCY3890 (swh3-ts16), transformed with the 2?m plasmid YEp352 [HTL1], or YEp352 alone, was streaked with the isogenic wild-type strain
W303-1A (RSC8) onto YEPD and incubated at 37°C for 3 days.
8170ROMEO ET AL.MOL. CELL. BIOL.

Page 7

ization (Fig. 5). Similar results were obtained when Htl13xHA
was expressed from a centromeric plasmid but with a less
intense signal (data not shown).
Htl1 can associate with the RSC complex. The genetic con-
nections between HTL1 and components of the RSC complex
established above, combined with the nuclear localization of
Htl1, suggested that this protein might physically associate with
the RSC complex. To explore this possibility, we tested for in
vivo association of Htl1 with the Sth1 ATPase subunit of RSC
(11). For this purpose, Htl1 was fused at its N terminus to a
Flag epitope (FlagHTL1) and overexpressed in yeast cells
(YBC928) from the MET25 promoter. Immunoprecipitation of
FlagHtl1 from extracts coprecipitated a small proportion of the
Sth1 (Fig. 6A, top blot). The observed coprecipitation reflects
a true association ofFlagHtl1 with RSC, because Sth1 was not
detected in the absence of eitherFlagHtl1 or anti-Flag antibod-
ies. Interestingly, Sth1 was also detected in the supernatant
fraction (Fig. 6A, top blot) that had been immunodepleted of
overexpressedFlagHtl1 (Fig. 6A, bottom blot), suggesting that
Htl1 is associated with only a subset of RSC complexes. In a
similar immunoprecipitation ofFlagHtl1, either Rsc3 or its mu-
tant form Rsc3-3 was detected with the coprecipitated Sth1
(Fig. 6B), indicating that Htl1 can associate with the form of
RSC that contains Rsc3. In reciprocal immunoprecipitations,
overexpressedFlagHtl1 was weakly detected in Sth1 immune
complexes (Fig. 6C), supporting the conclusion that Htl1 can
associate with a fraction of the RSC complex in vivo. We could
not detect Htl13xHAexpressed at endogenous levels from its
own promoter in complex with Sth1, suggesting that the RSC-
associated fraction may be small.
DISCUSSION
The RSC chromatin remodeling complex has emerged as an
important transcriptional regulator of many genes, including
those important for cell wall biogenesis, NDP, ribosome bio-
genesis, and carbohydrate metabolism (1, 9, 34). Additionally,
RSC is essential for progression of the cell cycle through G2/M.
Here, we report the association of Htl1 with RSC complex and
functional links between RSC/Htl1 and Pkc1 signaling. HTL1
is a 78-amino-acid protein required for growth at 37°C and for
maintenance of fertility and ploidy at permissive temperatures
(25). Here, we isolated HTL1 in a genetic screen for additive
growth defects with a conditional mutation in PKC1 (stt1-1).
We now demonstrate that HTL1 is required for cell cycle
progression through the G2/M transition at temperatures
FIG. 5. Htl13xHAresides in the nucleus. Wild-type diploid cells
(strain 1788), harboring HTL13xHAon a 2?m plasmid were grown to
mid-log phase in YEPD, fixed, and subjected to indirect immunoflu-
orescence microscopy with mouse monoclonal anti-HA antibody
(12CA5) (?-HA) and fluorescein isothiocyanate (FITC)-conjugated
secondary antibody. Nuclear DNA was visualized with DAPI. The
no-antibody (?Ab) control cells (bottom) were treated with secondary
antibody only.
FIG. 6.
Extracts were prepared from yeast strain YBC928 bearing either
pMET-Flag-HTL1 or vector. Immunoprecipitations were conducted by
incubating 300 ?g of extract with either anti-Flag M2 affinity gel
(?Flag) (A) or anti-Sth1 (?Sth1) conjugated to protein A beads (C).
Control beads with no antibodies were included for both. Samples
were eluted either with Flag peptide (A) or with SDS sample buffer
(C), separated by SDS-polyacrylamide gel electrophoresis, and sub-
jected to immunoblot analysis with either anti-Sth1 or anti-Flag M2
antisera. Load (L) (60 ?g), immunodepleted supernatant (S) (18%),
and eluate (E) (50%) samples are shown. (B) Extracts were prepared
from yeast strains YBC843 (RSC3) and YBC906 (rsc3-3) bearing
pMET-Flag-HTL1, andFlagHtl1 was immunoprecipitated and eluted as
described above for panel A. Samples were separated by SDS-poly-
acrylamide gel electrophoresis and subjected to immunoblot analysis
with both anti-Sth1 and anti-Rsc3 antisera. Load (L) (60 ?g) and
eluate (E) (50%) samples are shown. The positions of molecular size
standards (in kilodaltons) are shown to the left of the blots.
FlagHtl1 associates with the RSC complex in vivo. (A and C)
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above 33°C and that the Htl1 protein can associate with the
RSC complex. We propose that Pkc1 and RSC are coregula-
tors of the G2/M transition.
Htl1 can associate with the RSC complex. We present four
lines of evidence supporting the conclusion that Htl1 interacts
physically and functionally with the RSC complex. First, at
restrictive temperatures, htl1 null mutants exhibit a uniform
cell cycle arrest in G2/M that is similar to that of strains with
conditional mutations in some essential components of the
RSC complex (e.g., alleles of STH1, SFH1, RSC3, and RSC9 [1,
7, 9, 45]). For reasons that are not clear, the cell cycle-specific
arrest of an htl1 null mutant was not observed in a previous
study (25). Strain background differences may explain this dis-
parity, as may the possible acquisition of suppressor mutations.
Second, conditional rsc3 haploid strains accumulate cells with
4N DNA content (1), as was shown for htl1? haploids (25).
Third, multicopy suppression experiments revealed a genetic
interaction between HTL1 and RSC components. Specifically,
overexpression of RSC3, an essential DNA-binding component
of the RSC complex, suppressed the growth defect of an htl1
null mutant. Additionally, overexpression of HTL1 suppressed
the growth defect of a conditional rsc8 mutant. The observa-
tion that an increased dose of HTL1 could not suppress the
growth defect of any of three conditional rsc3 mutations sug-
gests that its overexpression can compensate for some RSC
defects, but not others. Fourth, Htl1 is a nuclear protein that
when overexpressed was found to associate in vivo with known
RSC complex components Rsc3 and Sth1.
A recent quantitative study of proteins associated with the
general transcription factor TFIID (41), which is a multisub-
unit complex comprised of the TATA-binding protein (TBP)
and 14 TBP-associated factors (TAFs), revealed that all known
RSC components were among the constellation of proteins
that displayed preferential association with TBP (compared
with TAFs). Htl1 was among the other, non-RSC proteins
identified in this population. Our results, which indicate that
Htl1 can associate with RSC, provide an explanation for the
reported association of Htl1 with TBP.
Why was Htl1 not identified in previous characterizations of
RSC components? The most likely explanation is that Htl1
appears to exist in substoichiometric amounts within the RSC
complex. We found that cell extracts that were immunode-
pleted of overexpressedFlagHtl1 still possessed some of the
Sth1 subunit. Moreover, it was possible to detectFlagHtl1 as-
sociated with immunoprecipitates of Sth1 only when the
former was greatly overproduced. This may be significant in
light of the observation that RSC exists in forms that either
possess or are devoid of Rsc3/30 (5). The genetic interaction
we observed between HTL1 and RSC3 may reflect a specific
association of Htl1 with the form of RSC that contains Rsc3/
30. The Rsc3/30 proteins exist as a heterodimeric complex that
can form outside of the RSC complex (1). Interestingly, al-
though these closely related components serve overlapping
roles, they may act antagonistically for certain functions. Our
finding that the htl1 null growth defect was suppressed by
overexpression of RSC3, but not RSC30, underscores the func-
tional differences between these components.
Interactions between the RSC complex and Pkc1. Recent
studies have raised the possibility that RSC function is regu-
lated by Pkc1. First, a genetic screen for multicopy suppressors
of the growth defect of a temperature-sensitive mutation in
STH1/NPS1, which encodes the DNA-dependent ATPase of
the RSC complex, yielded upstream components of the cell
wall integrity pathway, including PKC1 (14). Overexpression or
mutational activation of elements of the MAP kinase cascade
controlled by Pkc1 failed to suppress sth1, prompting these
investigators to suggest that the observed effect of Pkc1 is not
mediated by the MAP kinase pathway. Additive growth defects
between conditional alleles of STH1 (nps1-105) and PKC1
(stt1-1) also support a functional connection to RSC (14). A
recent study by Chai et al. (7a) confirmed these findings using
another allele of STH1 (sth1-3) and also demonstrated additive
growth defects between sth1-3 and a null mutation in the cell
surface sensor for Pkc1 activation encoded by WSC1. Second,
mutation of either RSC3 or RSC30 alters the expression of
several genes implicated in cell wall biogenesis, and the sensi-
tivity to caffeine and formamide displayed by conditional RSC3
mutants is suppressed by overexpression of PKC1 (1). More-
over, rsc3 mutations are lethal in combination with a pkc1 null
allele (even in the presence of osmotic support), but not a bck1
null allele. Collectively, these observations strongly suggest a
functional link between Pkc1 and RSC.
Loss of Pkc1 function results in cell lysis resulting from a
deficit in cell wall construction (29, 30). However, loss of func-
tion in the MAP kinase cascade regulated by Pkc1 results in
cell lysis only at elevated temperatures (30), indicating that
Pkc1 regulates at least one other pathway that contributes to
cell wall integrity. We found that loss of HTL1 function was
lethal in the presence of pkc1 mutations but resulted in only
mild defect additivity with a bck1 null allele, similar to the
behavior of other rsc mutants. Although the observed defect
additivity suggests that htl1 mutants may suffer a deficiency in
cell wall biogenesis, we observed no evidence of cell lysis at
restrictive temperatures and were unable to detect a cell wall
defect through tests for hypersensitivity to cell wall stresses.
An interesting alternative explanation for the observed de-
fect additivity between pkc1 and htl1 invokes a role for Pkc1 in
passage through G2/M. Depletion of Pkc1 results in cell lysis at
the bud tip during a distinct period in the cell cycle, previously
interpreted to reflect the point at which the cell is most sensi-
tive to cell wall stress (30). Such cells die uniformly with single,
postreplication nuclei and small buds (32). This is also the case
for conditional alleles of pkc1 (29). Hosotani et al. (14) re-
cently extended these observations, finding that a conditional
pkc1 mutant (stt1-1) undergoes a G2/M delay at permissive
temperatures. These investigators suggested that Pkc1 might
have a cell cycle function that is independent of cell wall
metabolism. Because we isolated an htl1 mutant in a synthetic
lethal screen with stt1-1, our finding that an htl1 null mutant
also displays a G2/M delay at permissive temperatures suggests
that a double pkc1 htl1 mutant may succumb to a G2/M block.
A cell cycle block could explain our inability to rescue this
double mutant by osmotic support.
Models for RSC/Htl1 function and Pkc1 cooperativity in cell
wall biogenesis and cell cycle progression. The striking defect
additivity observed between pkc1? and either rsc3 alleles (1) or
htl1? suggests that cell viability requires either intact Pkc1
function or intact RSC function. However, this relationship
(and other data) does not establish whether these factors func-
tion together in a single pathway or coordinately in parallel
8172ROMEO ET AL. MOL. CELL. BIOL.

Page 9

pathways (Fig. 7). In considering a single pathway, Pkc1 could
phosphorylate RSC components and alter RSC function at loci
important for cell wall biogenesis and G2/M progression.
Clearly, Pkc1 regulation of RSC would have to be limited to a
subset of RSC functions, because null mutations in most RSC
components are lethal even in the presence of osmotic support
(pkc1? mutants are viable under these conditions). Moreover,
RSC occupies hundreds of genes not related to Pkc1 function.
However, to date, we have been unable to detect phosphory-
lation of purified RSC by Pkc1 in vitro (unpublished observa-
tions), though we have not ruled out the possibility that RSC is
a Pkc1 target in vivo. A second model equally consistent with
our data are that Pkc1 and RSC act coordinately in parallel
pathways to regulate cell wall biogenesis and passage through
G2/M. In this case, RSC components are not direct targets of
Pkc1. Instead, the functions of both of these factors are re-
quired for proper expression of genes involved in cell wall
biogenesis and passage through the G2/M transition. In either
case, our data strongly suggest that Htl1 assists RSC primarily
for proper passage through G2/M phase (Fig. 7), as htl1 null
mutants phenotypically copy the G2/M cell cycle block ob-
served in certain rsc mutants but lack the cell wall phenotypes
associated with rsc3 mutations.
ACKNOWLEDGMENTS
We thank Takahiro Negishi for assistance with htl1 suppressor
screening and Southern blotting, Forrest Spencer for providing the
genomic library, and Marian Carlson for providing yeast strains.
This work was supported in part by NIH grants GM48533 to D.E.L.
and GM60415 to B.R.C. Training grant 5T32CA09110 supported
M.J.R. and A.K.S. Training grant T32GM07464 supported M.L.A.-
H. B.R.C. is an Assistant Investigator with the Howard Hughes Med-
ical Institute.
REFERENCES
1. Angus-Hill, M. L., A. Schlichter, D. Roberts, H. Erdjument-Bromage, P.
Tempst, and B. R. Cairns. 2001. A Rsc3/Rsc30 zinc cluster dimer reveals
novel roles for the chromatin remodeler RSC in gene expression and cell
cycle control. Mol. Cell 7:741–751.
2. Armstrong, J. A., J. J. Bieker, and B. M. Emerson. 1998. A SWI/SNF-related
chromatin remodeling complex, E-RC1, is required for tissue-specific tran-
scriptional regulation by EKLF in vitro. Cell 95:93–104.
3. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for
mutants lacking orotidine-5?-phosphate decarboxylase activity in yeast:
5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345–346.
4. Cairns, B. R. 1998. Chromatin remodeling machines: similar motors, ulterior
motives. Trends Biochem. Sci. 23:20–25.
5. Cairns, B. R., Y. Lorch, Y. Li, M. Zhang, L. Lacomis, H. Erdjument-Bro-
mage, P. Tempst, J. Du, B. Laurent, and R. D. Kornberg. 1996. RSC, an
essential, abundant chromatin-remodeling complex. Cell 87:1249–1260.
6. Cairns, B. R., A. Schlichter, H. Erdjument-Bromage, P. Tempst, R. D.
Kornberg, and F. Winston. 1999. Two functionally distinct forms of the RSC
nucleosome-remodeling complex, containing essential AT hook, BAH, and
bromodomains. Mol. Cell 4:715–723.
7. Cao, Y., B. R. Cairns, R. D. Kornberg, and B. C. Laurent. 1997. Sfh1p, a
component of a novel chromatin-remodeling complex, is required for cell
cycle progression. Mol. Cell. Biol. 17:3323–3334.
7a.Chai, B., J. Hsu, J. Du, and B. C. Laurent. 2002. Yeast RSC function is
required for organization of the cellular cytoskeleton via an alternative PKC1
pathway. Genetics 161:575–584.
8. Costigan, C., S. Gehrung, and M. Snyder. 1992. A synthetic lethal screen
identifies SLK1, a novel protein kinase homolog implicated in yeast cell
morphogenesis and cell growth. Mol. Cell. Biol. 12:1162–1178.
9. Damelin, M., I. Simon, T. I. Moy, B. Wilson, S. Komili, P. Tempst, F. P.
Roth, R. A. Young, B. R. Cairns, and P. A. Silver. 2002. The genome-wide
localization of Rsc9, a component of the RSC chromatin-remodeling com-
plex, changes in response to stress. Mol. Cell 9:563–573.
10. Dodou, E., and R. Treisman. 1997. The Saccharomyces cerevisiae MADS-box
transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein
kinase pathway. Mol. Cell. Biol. 17:1848–1859.
11. Du, J., I. Nasir, B. K. Benton, M. P. Kladde, and B. C. Laurent. 1998. Sth1p,
a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essential ATPase in
RSC and differs from Snf/Swi in its interactions with histones and chromatin-
associated proteins. Genetics 150:987–1005.
12. Gray, J. V., J. P. Ogas, Y. Kamada, M. Stone, D. E. Levin, and I. Herskowitz.
1997. A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae
in bud emergence and identification of a putative upstream regulator.
EMBO J. 16:4924–4937.
13. Hill, J. E., A. M. Muers, T. J. Koerner, and A. Tzagoloff. 1986. Yeast/E. coli
shuttle vectors with multiple unique restriction sites. Yeast 2:163–167.
14. Hosotani, T., H. Koyama, M. Uchino, T. Miyakawa, and E. Tsuchiya. 2001.
PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, partici-
pates in microtubule function through the yeast EB1 homologue, BIM1.
Genes Cells 6:775–788.
15. Irie, K., M. Takase, K. S. Lee, D. E. Levin, H. Araki, K. Matsumoto, and Y.
Oshima. 1993. MKK1 and MKK2, which encode Saccharomyces cerevisiae
mitogen-activated protein kinase-kinase homologs, function in the pathway
mediated by protein kinase C. Mol. Cell. Biol. 13:3076–3083.
16. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of
intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168.
17. Jacoby, J. J., S. M. Nilius, and J. J. Heinisch. 1998. A screen for upstream
components of the yeast protein kinase C signal transduction pathway iden-
tifies the product of the SLG1 gene. Mol. Gen. Genet. 258:148–155.
18. Jung, U. S., and D. E. Levin. 1999. Genome-wide analysis of gene expression
regulated by the yeast cell wall integrity signaling pathway. Mol. Microbiol.
34:1049–1057.
19. Kadonaga, J. T. 1998. Eukaryotic transcription: an interlaced network of
transcription factors and chromatin-modifying machines. Cell 92:307–313.
20. Kamada, Y., U. S. Jung, J. Piotrowski, and D. E. Levin. 1995. The protein
kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae medi-
ates a novel aspect of the heat shock response. Genes Dev. 9:1559–1571.
21. Kamada, Y., H. Qadota, C. P. Python, Y. Anraku, Y. Ohya, and D. E. Levin.
1996. Activation of yeast protein kinase C by Rho1 GTPase. J. Biol. Chem.
271:9193–9195.
22. Ketela, T., R. Green, and H. Bussey. 1999. Saccharomyces cerevisiae Mid2p is
a potential cell wall stress sensor and upstream activator of the PKC1-MPK1
cell integrity pathway. J. Bacteriol. 181:3330–3340.
23. Kingston, R. E., and G. J. Narlikar. 1999. ATP-dependent remodeling and
acetylation as regulators of chromatin fluidity. Genes Dev. 13:2339–2352.
24. Kornberg, R. D., and Y. Lorch. 1999. Chromatin-modifying and -remodeling
complexes. Curr. Opin. Genet. Dev. 9:148–151.
FIG. 7. Models for the interaction of Pkc1 with RSC in the regulation of cell wall biogenesis and G2/M.
VOL. 22, 2002Htl1 INTERACTS WITH RSC COMPLEX IN S. CEREVISIAE8173

Page 10

25. Lanzuolo, C., S. Ederle, A. Pollice, F. Russo, A. Storlazzi, and J. F. Pulitzer.
2001. The HTL1 gene (YCR020W-b) of Saccharomyces cerevisiae is neces-
sary for growth at 37°C, and for the conservation of chromosome stability
and fertility. Yeast 18:1317–1330.
26. Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsu-
moto, and D. E. Levin. 1993. A yeast mitogen-activated protein kinase
homolog (Mpk1) mediates signaling by protein kinase C. Mol. Cell. Biol.
13:3067–3075.
27. Lee, K. S., and D. E. Levin. 1992. Dominant mutations in a gene encoding a
putative protein kinase (BCK1) bypass the requirement for a Saccharomyces
cerevisiae protein kinase C homolog. Mol. Cell. Biol. 12:172–182.
28. Leibowitz, M. J., and R. B. Wickner. 1978. Pet18: a chromosomal gene
required for cell growth and for the maintenance of mitochondrial DNA and
the killer plasmid of yeast. Mol. Gen. Genet. 165:115–121.
29. Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae
PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol.
116:1221–1229.
30. Levin, D. E., B. Bowers, C. Chen, Y. Kamada, and M. Watanabe. 1994.
Dissecting the protein kinase C/MAP kinase signaling pathway of Saccharo-
myces cerevisiae. Cell. Mol. Biol. Res. 40:229–239.
31. Levin, D. E., and B. Errede. 1995. The proliferation of MAP kinase signaling
pathways in yeast. Curr. Opin. Cell Biol. 7:197–202.
32. Levin, D. E., F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner. 1990.
A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell
cycle. Cell 62:213–224.
33. Martin, H., J. Arroyo, M. Sanchez, M. Molina, and C. Nombela. 1993.
Activity of the yeast MAP kinase homolog Slt2 is critically required for cell
integrity at 37°C. Mol. Gen. Genet. 241:177–184.
34. Ng, H. H., F. Robert, R. A. Young, and K. Struhl. 2002. Genome-wide
location and regulated recruitment of the RSC nucleosome-remodeling
complex. Genes Dev. 16:806–819.
35. Nonaka, H., K. Tanaka, H. Hirano, T. Fujiwara, H. Kohno, M. Umikawa, A.
Mino, and Y. Takai. 1995. A downstream target of RHO1 small GTP-
binding protein is PKC1, a homolog of protein kinase C, which leads to
activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J.
14:5931–5938.
36. Ozaki, K., K. Tanaka, H. Imamura, T. Hihara, T. Kamayema, H. Nonaka, H.
Hirano, Y. Matsuura, and Y. Takai. 1996. Rom1p and Rom2p are small
GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP-binding
protein in Saccharomyces cerevisiae. EMBO J. 15:2196–2207.
37. Paravicini, G., M. Cooper, L. Friedli, D. J. Smith, J.-L. Carpentier, L. S.
Klig, and M. A. Payton. 1992. The osmotic integrity of the yeast cell requires
a functional PKC1 gene product. Mol. Cell. Biol. 12:4896–4905.
38. Peterson, C. L., and J. L. Workman. 2000. Promoter targeting and chromatin
remodeling by the SWI/SNF complex. Curr. Opin. Genet. Dev. 10:187–192.
39. Rajavel, M., B. Philip, B. M. Buehrer, B. Errede, and D. E. Levin. 1999. Mid2
is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.
Mol. Cell. Biol. 19:3969–3976.
40. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
41. Sanders, S. L., J. Jennings, A. Canutescu, A. J. Link, and P. A. Weil. 2002.
Proteomics of the eukaryotic transcription machinery: identification of pro-
teins associated with components of yeast TFIID by multidimensional mass
spectrometry. Mol. Cell. Biol. 22:4723–4738.
42. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast
host strains designed for efficient manipulation of DNA in Saccharomyces
cerevisiae. Genetics 122:19–27.
43. Toh-e, A., and Y. Sahashi. 1985. The PET18 locus of Saccharomyces cerevi-
siae: a complex locus containing multiple genes. Yeast 1:159–171.
44. Treich, I., L. Ho, and M. Carlson. 1998. Direct interaction between Rsc6 and
Rsc8/Swh3, two proteins that are conserved in SWI/SNF-related complexes.
Nucleic Acids Res. 26:3739–3745.
45. Tsuchiya, E., M. Uno, A. Kiguchi, K. Masuoka, Y. Kanemori, S. Okabe, and
T. Mikayawa. 1992. The Saccharomyces cerevisiae NPS1 gene, a novel CDC
gene which encodes a 160 kDa nuclear protein involved in G2phase control.
EMBO J. 11:4017–4026.
46. Verna, J., A. Lodder, K. Lee, A. Vagts, and R. Ballester. 1997. A family of
genes required for the maintenance of cell wall integrity and for the stress
response in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 94:13804–13809.
47. Watanabe, Y., G. Takaesu, M. Hagiwara, K. Irie, and K. Matsumoto. 1997.
Characterization of a serum response factor-like protein in Saccharomyces
cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1
(Slt2) mitogen-activated protein kinase pathway. Mol. Cell. Biol. 17:2615–
2623.
48. Wu, C., T. Tsukiyama, D. Gdula, P. Georgel, M. Martinez-Balbas, G. Mi-
zuguchi, V. Ossipow, R. Sanaltzopoulos, and H. M. Wang. 1998. ATP-
remodeling of chromatin. Cold Spring Harbor Symp. Quant. Biol. 63:525–
534.
49. Yoshida, S., E. Ikeda, I. Uno, and H. Mitsuzawa. 1992. Characterization of
a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces
cerevisiae: STT1 is allelic to PKC1. Mol. Gen. Genet. 231:337–344.
8174 ROMEO ET AL.MOL. CELL. BIOL.