Yeast RSC Function Is Required for Organization of the Cellular Cytoskeleton via an Alternative PKC1 Pathway

Corresponding author: Brehon C. Laurent, SUNY Downstate Medical Center, 450 Clarkson Ave., Box 44, Brooklyn, NY 11203., blaurent{at}netmail.hscbklyn.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

RSC is a 15-protein ATP-dependent chromatin-remodeling complex related to Snf-Swi, the prototypical ATP-dependent nucleosome remodeler in budding yeast. Despite insight into the mechanism by which purified RSC remodels nucleosomes, little is known about the chromosomal targets or cellular pathways in which RSC acts. To better understand the cellular function of RSC, a screen was undertaken for gene dosage suppressors of sth1-3ts, a temperature-sensitive mutation in STH1, which encodes the essential ATPase subunit. Slg1p and Mid2p, two type I transmembrane stress sensors of cell wall integrity that function upstream of protein kinase C (Pkc1p), were identified as multicopy suppressors of sth1-3ts cells. Although the sth1-3ts mutant exhibits defects characteristic of PKC1 pathway mutants (caffeine and staurosporine sensitivities and an osmoremedial phenotype), only upstream components and not downstream effectors of the PKC1-MAP kinase pathway can suppress defects conferred by sth1-3ts, suggesting that RSC functions in an alternative PKC1-dependent pathway. Moreover, sth1-3ts cells display defects in actin cytoskeletal rearrangements and are hypersensitive to the microtubule depolymerizing drug, TBZ; both of these defects can be corrected by the high-copy suppressors. Together, these data reveal an important functional connection between the RSC remodeler and PKC1-dependent signaling in regulating the cellular architecture.

CHROMOSOMES are the substrates for many essential nuclear processes including DNA replication, repair, recombination, and transcription. These events must be coordinated with the dynamic changes in chromosome structure and cell morphology that occur throughout the cell cycle. Although chromatin interferes with several of these DNA-dependent cellular functions, chromatin-remodeling complexes are able to alter accessibility to DNA. Two general classes of remodeling enzymes have been identified. The first includes ATP-dependent chromatin remodelers, which use the energy released from ATP hydrolysis to disrupt histone-DNA interactions (for reviews see KINGSTON and NARLIKAR 1999 ; VIGNALI et al. 2000 ). The second includes enzymes that covalently modify histones by adding ubiquitin, acetyl, methyl, or phosphate groups (for reviews see STRAHL and ALLIS 2000 ; BERGER 2001 ; BERGER and FELSENFELD 2001 ; JENUWEIN and ALLIS 2001 ; ROTH et al. 2001 ). These two types of enzyme function coordinately to regulate transcription at some promoters (see FRY and PETERSON 2001 ).

In addition to yeast Snf-Swi, the prototypical ATP-dependent chromatin-remodeling complex, the SWI/SNF subfamily also includes the yeast RSC (for remodels the structure of chromatin), Drosophila brahma, and human BRG-1 and hbrm complexes (COTE et al. 1994 ; KWON et al. 1994 ; CAIRNS et al. 1996 ; PAPOULAS et al. 1998 ). RSC is an abundant 1–2 MD complex composed of 15 proteins (CAIRNS et al. 1996 ). Like all ATP-dependent remodelers, RSC contains a Snf2p/Swi2p-related ATPase subunit, Sth1p. However, despite its similarity to the yeast Snf-Swi complex, RSC is required for cell viability. The G2/M arrest conferred by four temperature-sensitive (ts) rsc mutations suggests a requirement for RSC function in cell cycle progression (TSUCHIYA et al. 1992 ; CAO et al. 1997 ; DU et al. 1998 ; ANGUS-HILL et al. 2001 ). RSC function has also been implicated in transcription (CAO et al. 1997 ; CAIRNS et al. 1998 , CAIRNS et al. 1999 ; MOREIRA and HOLMBERG 1999 ; YUKAWA et al. 1999 ; ANGUS-HILL et al. 2001 ). Interestingly, RSC shares functional and structural similarities with human SWI/SNF-B (NIE et al. 2000 ; VIGNALI et al. 2000 ).

Cellular signaling plays an important role in relaying extracellular information to intracellular pathways that impact on nuclear function. Protein kinase C (PKC)/ras and Ca²⁺-mediated membrane signaling has been shown to regulate hSWI/SNF chromatin remodeling during T-lymphocyte activation (ZHAO et al. 1998 ). Mammalian PKC pathways regulate diverse cellular processes including cell cycle progression, differentiation, growth control, and tumor promotion (LIVNEH and FISHMAN 1997 ). Similarly, the single isozyme of protein kinase C in Saccharomyces cerevisiae controls a variety of cellular processes such as cell cycle progression, mating, nutrient sensing, and the structural organization of the cytoskeleton (HEINISCH et al. 1999 ). Pkc1p is activated by a small GTPase of the Rho family, Rho1p, which receives upstream signals from the Slg1p and Mid2p transmembrane sensors (PHILIP and LEVIN 2001 ). The downstream PKC1-mitogen-activated protein kinase (MAPK) cascade, consisting of Bck1p-Mkk1p/Mkk2p-Mpk1p, phosphorylates transcription factors that regulate cell wall and cytoskeleton integrity in polarized cell growth (HEINISCH et al. 1999 ). Although less well defined, several studies report evidence for pathways that branch at Pkc1p (DELLEY and HALL 1999 ; KETELA et al. 1999 ; ANDREWS and STARK 2000 ; LI et al. 2000 ; NANDURI and TARTAKOFF 2001 ).

Despite progress in characterizing the remodeling mechanism of purified RSC, the cellular processes in which RSC functions and the chromosomal targets of RSC remain largely unknown. In this study, we carried out a genetic screen for multicopy suppressors of the sth1-3ts cell cycle mutation to identify targets that would define the pathways in which Sth1p and RSC are involved. The identification of two related cell wall integrity sensors that function upstream of protein kinase C (PKC1) as suppressors revealed a functional connection to the PKC1-dependent signal transduction pathway. Together, analysis of the rsc mutant phenotypes and the identities of suppressors and profiles of suppression suggest that RSC function is required for regulating the cellular cytoskeleton.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains, media, and standard methods:
All S. cerevisiae strains were isogenic to S288c and are listed in Table 1. Yeast cultures were grown in rich media, consisting of yeast extract, peptone, and 2% dextrose (YPD), or selective synthetic complete (SC) media containing 2% dextrose (GUTHRIE and FINK 1991 ). Yeast transformations were carried out by the lithium acetate procedure (ITO et al. 1983 ). The plasmids and oligonucleotides used in this study are listed in Table 2 and Table 3, respectively.

A one-step PCR gene disruption method was used to generate mid2, slg1, mid2 sth1-3ts, or slg1 sth1-3ts strains as described (BAUDIN et al. 1993 ). The coding sequences of SLG1 or MID2 were replaced with the HIS3 gene amplified from pRS313 using oligos SLG1-up and SLG1-down or MID2-up and MID2-down, respectively. The resulting PCR products, containing the HIS3 gene flanked by 40 nucleotides of the 5' and 3' noncoding sequences of SLG1 or MID2 were transformed into the wild-type STH1 (BLY1) and sth1-3ts (BLY49) strains to generate BLY452 and BLY453 or BLY454 and BLY455, respectively. Deletion of SLG1 or MID2 was verified by PCR analysis using primers 100 bp upstream of the SLG1 or MID2 genes and within the HIS3 gene.

Identification of high-copy extragenic suppressors of sth1-3ts:
sth1-3ts (BLY49) cells were transformed with a genomic library cloned into the YEp24 multicopy vector (CARLSON and BOTSTEIN 1982 ) and selected for uracil prototrophy on SC-Ura medium at 30° for 2 days. Approximately 75,000 independent Ura⁺ transformants were subsequently replica printed to SC-Ura and grown at 37° for 5 days. Twenty-eight colonies that conferred growth at 37° were isolated. Library clones were recovered from 25 of the yeast strains and retested for the suppression phenotype. Restriction endonuclease digestion of the plasmid DNAs recovered from the yeast strains indicated that 13 plasmids contained STH1 sequences; the remaining 12 clones represented suppressors in eight different groups. To identify the genes responsible for the suppression phenotype, candidate genes encoded by the suppressor plasmids were amplified by PCR, subcloned into YEp24, and retested for suppression of the sth1-3ts ts phenotype. The two strongest suppressors, pMID2 and pSLG1, were pursued in this study.

Flow cytometry:
Cells were grown in SC selective medium at 30° to midlog phase, split, and diluted into prewarmed 30° or 37° SC-Ura media at concentrations of 4 x 10⁶ cells/ml. Shifted cells were fixed in 70% ethanol, stained with propidium iodide, and their fluorescence intensities were measured as described (DU et al. 1998 ).

Actin staining:
Midlog phase cells carrying plasmids grown in SC selective medium at 25° were diluted and shifted into 37° prewarmed SC media. Shifted cells were fixed in 3.7% formaldehyde in 100 mM potassium phosphate buffer (pH 6.5) and stained with 1.2 units of rhodamine phalloidin (Molecular Probes, Eugene, OR) for 2 hr at room temperature (ADAMS and PRINGLE 1984 ). Stained cells were washed three to five times in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄). Fluorescence images were acquired using a KAI-2092M cooled CCD camera (Diagnostic Instruments) with Spot software on a Nikon Labophoto-II microscope.

RNA analysis:
Total RNA was prepared by glass bead disruption as described previously (LAURENT et al. 1990 ). ³²P-labeled hybridization probes were generated by PCR amplification using primer pairs MID2 5' and MID2 3', SLG1 5' and SLG1 3', ACT1-up and ACT1-down, and 5upTUB1 and 3dwTUB1 as described (DU et al. 1998 ). RNA levels were quantified with ImageQuant software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of SLG1/WSC1 and MID2 as multicopy suppressors of the sth1-3ts mutant:
To better understand the cellular function of the RSC complex, we sought to identify gene dosage suppressors of sth1-3ts, a temperature-sensitive mutation in STH1, which encodes the essential ATPase subunit. sth1-3ts mutants arrest in the G2/M phase of the cell division cycle at the nonpermissive temperature (37°) and exhibit a variety of cellular defects (DU et al. 1998 ). We reasoned that the isolation of gene suppressors that could restore growth of sth1-3ts cells at 37° would identify factors that interact with RSC and/or uncover the cellular processes that require RSC function.

To screen for multicopy suppressors, the sth1-3ts strain was transformed with a yeast genomic DNA library cloned into YEp24 (CARLSON and BOTSTEIN 1982 ). In addition to the expected wild-type STH1 gene, we isolated several genes that when overexpressed rescued the ts lethal phenotype of the mutant at 37°. Here we report on the two strongest suppressors. DNA sequence analysis identified a single clone containing SLG1/WSC1/HCS77 (synthetic lethal with Gap 1/cell wall integrity and stress response component 1) and three independent clones containing MID2 (mating pheromone-induced death 2; Fig 1). The encoded proteins, Slg1p and Mid2p, are plasma membrane-associated proteins with overlapping functions as upstream cell wall stress sensors that activate the PKC1-MAP kinase pathway (GRAY et al. 1997 ; VERNA et al. 1997 ; JACOBY et al. 1998 ; RAJAVEL et al. 1999 ; STIRLING and STARK 2000 ).

View larger version (50K): In this window In a new window Download PPT slide   Figure 1. High-copy MID2 or SLG1 suppresses the temperature-sensitive growth of sth1-3ts mutants. Tenfold serial dilutions of midlogarithmic phase sth1-3ts (BLY49) cells harboring pBL50 (STH1), YEp24 (vector), pMID2 (MID2), or pSLG1 (SLG1) were plated onto SC plates to determine growth at permissive (25°) or nonpermissive (37°) temperatures. The plates were photographed after 3–4 days of growth.

Interestingly, overexpression of either SLG1 or MID2 can also suppress the temperature sensitivity conferred by a different STH1 allele, sth1-1ts (data not shown). However, neither gene can suppress sfh1-1::HIS3, a mutant allele of a second well-conserved RSC component, Sfh1p. High-copy SLG1 or MID2 also failed to suppress ssn20-1, a mutant allele of a chromatin-associated protein, Spt6p (data not shown). These results indicate that the sth1-3ts suppressors are gene specific.

sth1-3ts mutants display phenotypes characteristic of PKC1 pathway mutants:
Pkc1p plays a key regulatory role in maintaining the integrity of the cytoskeleton and cell wall and links extracellular signals to intracellular responses (LEVIN et al. 1990 ; LEVIN and BARTLETT- HEUBUSCH 1992 ; PARAVICINI et al. 1992 ). Identification of upstream components of the PKC1-MAP kinase components as suppressors suggests a link between RSC function and the PKC1 pathway. To further establish this connection, we examined whether sth1-3ts mutants show cell wall integrity or other PKC1 pathway defects. Loss-of-function mutations in components of the PKC1 signaling pathway result in osmotic fragility due to defects in cell wall biogenesis that can be suppressed by the presence of 1 M sorbitol, an osmotic stabilizer (LEVIN and BARTLETT-HEUBUSCH 1992 ; PARAVICINI et al. 1992 ; ROEMER et al. 1994 ). We found that addition of 1 M sorbitol suppressed the temperature sensitivity of sth1-3ts, suggesting that sth1-3ts confers osmotic fragility similar to PKC1 pathway mutants (Fig 2A; LEVIN and BARTLETT-HEUBUSCH 1992 ; GRAY et al. 1997 ).

View larger version (50K): In this window In a new window Download PPT slide   Figure 2. sth1-3ts mutants exhibit phenotypes characteristic of PKC1 pathway mutants. (A) sth1-3ts mutants show osmoremedial phenotypes. Threefold serial dilutions of log phase sth1-3ts (BLY49) and isogenic wild-type STH1 (BLY76) cells were spotted onto YPD plates or YPD plates containing 1 M sorbitol and growth was compared at 37°. (B) sth1-3ts cells are sensitive to caffeine and staurosporine and these sensitivities are suppressed by MID2 or SLG1. Threefold serial dilutions of log phase sth1-3ts (BLY49) cells carrying the indicated plasmids were plated onto YPD or YPD containing caffeine (6 mM) or staurosporine (5 µg/ml) and growth was compared at 30° after 3 days (caffeine) or 5 days (staurosporine).

Protein kinase C pathway mutants are also sensitive to caffeine, a phosphodiesterase inhibitor, and staurosporine, a protein kinase C inhibitor. Both caffeine and staurosporine caused nearly complete inhibition of growth of sth1-3ts cells at 30°. Moreover, these sensitivities were suppressed in cells overexpressing SLG1 or MID2 (Fig 2B). Similarly, defects caused by mutations in some PKC1 pathway components can be suppressed by overexpression of other components (MADDEN et al. 1997 ; HEINISCH et al. 1999 ). For example, the caffeine sensitivity of slg1 mutants can be suppressed by overexpression of PKC1 or RHO1 (KETELA et al. 1999 ). Thus, several key features of pkc1 pathway mutants are shared by sth1-3ts mutants.

STH1 interacts with upstream activators of the PKC1-dependent signaling pathway:
Signals from the Slg1p and Mid2p membrane sensors transmitted through Rom2p and Rho1p converge at the Pkc1p effector kinase. Thus, we reasoned that, if RSC acts through the PKC1 pathway, then high-copy PKC1 would also suppress the ts growth defects of sth1-3ts. Indeed, mutants expressing high-copy PKC1 were capable of growth at 37° and suppression was comparable to that by MID2 or SLG1 (Fig 3). Similarly, the intermediate transducers Rom2p (the GDP-GTP exchange factor that activates Rho1p) and Rho2p (a small GTP-binding protein in the Rho1p family believed to organize the actin cytoskeleton; MARCOUX et al. 2000 ; PHILIP and LEVIN 2001 ) also suppressed the ts growth defects of sth1-3ts, although Rho2p was consistently a weaker suppressor (Fig 3).

View larger version (87K): In this window In a new window Download PPT slide   Figure 3. PKC1 and its upstream activators, MID2, SLG1, ROM2, and RHO2, but not the downstream effector kinases MKK1 or MPK1, suppress the sth1-3ts temperature sensitivity. Tenfold serial dilutions of log phase sth1-3ts (BLY49) cells carrying the indicated high-copy plasmids were plated onto SC plates to determine growth at 25° or 37°. (RHO2 was tested because RHO1 in high copy can be toxic; ANDREWS and STARK 2000 .) All plates were photographed after 2–3 days of growth.

The best-characterized Pkc1p-dependent pathway activates the MAP kinase module Bck1p-Mkk1p-Mpk1p to regulate the cell wall integrity and actin cytoskeleton (BANUETT 1998 ; HEINISCH et al. 1999 ). Thus, we investigated whether these modulators could likewise suppress the sth1-3ts growth defects. In contrast to the upstream components, single-copy plasmid expression of BCK1-20, an activated allele of BCK1 that encodes the first component of the MAP kinase cascade (LEE and LEVIN 1992 ), or overexpression of the genes encoding the other downstream effector protein kinases Mkk1p, a MAPKK, and Mpk1p/Slt2p, the MAP kinase, failed to suppress sth1-3ts (Fig 3; data not shown). Therefore, the interaction between RSC and the upstream activators but not the downstream effectors of the PKC1-MAP kinase pathway suggests that RSC functions in alternate PKC1-mediated pathways.

Deletion of SLG1 but not MID2 is synthetically lethal with the sth1-3ts mutation:
To further characterize the genetic connection between RSC and the upstream PKC1 pathway, double mutants between sth1-3ts and deletions of the cell wall integrity signaling genes SLG1 or MID2 were constructed. The sth1-3ts mutation was found to be lethal at the semipermissive temperature (36°) in combination with the slg1 mutation (Fig 4). In contrast, the growth phenotypes of the sth1-3ts mid2 double mutants were no different from those of the single mutants. These data distinguish the genetic interactions between STH1 and the cell wall stress sensors SLG1 or MID2. Furthermore, the ability of MID2 to suppress sth1-3ts or slg1 single mutants but not an sth1-3ts slg1 double mutant (data not shown) suggests that Sth1p and Slg1p function in genetically redundant or parallel pathways and is in agreement with the previous study that SLG1 and MID2 have overlapping and distinct functions (STIRLING and STARK 2000 ).

View larger version (44K): In this window In a new window Download PPT slide   Figure 4. The sth1-3ts mutation interacts synthetically with the slg1 mutation. Wild-type (BLY1), sth1-3ts (BLY49), slg1 (BLY452), sth1-3ts slg1 (BLY453), mid2 (BLY454), and sth1-3ts mid2 (BLY455) cells were grown to midlog phase, 10-fold serially diluted, and spotted on YPD plates. Growth was compared at 30° and 36° after 2 days.

PKC1 and its upstream activators relieve the G2/M arrest of sth1-3ts cells:
The sth1-3ts mutation confers a G2/M cell cycle arrest (DU et al. 1998 ). The ability of SLG1, MID2, or PKC1 to suppress the ts growth defects caused by sth1-3ts prompted us to determine whether these genes improved growth by relieving these cells from G2/M arrest. Indeed, high-copy MID2, SLG1, and PKC1 could partially suppress the G2/M arrest phenotype of sth1-3ts as judged by flow cytometry (Fig 5; data not shown). As expected, downstream members of the PKC1-MAP kinase pathway failed to suppress the sth1-3ts cell cycle arrest (data not shown). This result suggests that the alternative PKC1-mediated signaling pathway participates in the cell cycle function of RSC.

View larger version (15K): In this window In a new window Download PPT slide   Figure 5. MID2 suppresses the sth1-3ts G2/M cell cycle arrest. sth1-3ts (BLY49) cells carrying pBL50 (STH1), YEp24 (vector), or pMID2 (MID2) were analyzed by flow cytometry 12 hr after being shifted from 30° to 37°.

Delocalization of the cortical actin cytoskeleton in sth1 mutants can be suppressed by high-copy MID2, SLG1, or PKC1:
The established role of Pkc1p in mediating the cell cycle-dependent organization of actin (HELLIWELL et al. 1998 ) implicates RSC in PKC1-dependent signaling pathways that control the cortical actin cytoskeleton. To assess the integrity and distribution of the actin cytoskeleton, we first visualized intracellular polymerized actin filaments by staining sth1-3ts cells carrying wild-type STH1 or vector alone grown at 25° or 37° with rhodamine-coupled phalloidin. In mutants carrying vector grown at permissive temperature (25°), both cortical actin patches and cable arrays were polarized in cells throughout the cell cycle in a pattern that was indistinguishable from that in sth1-3ts cells expressing wild-type STH1 at 25° or 37° (Fig 6A). In contrast, upon shifting mutants harboring vector alone to 37°, the majority of cells arrested with large, misshapen buds and actin patches were dispersed randomly throughout both mother and daughter cells. Most of these cells also contained unusual large actin clumps. Actin cables were not visible in these mutants, and in cells that had completed nuclear division, actin rings were never seen at the bud necks.

View larger version (153K): In this window In a new window Download PPT slide   Figure 6. sth1-3ts exhibits actin cytoskeletal defects that can be suppressed by MID2 or SLG1. (A) Cortical actin patch organization is aberrant in sth1-3ts (BLY49) cells shifted to 37° for 10 hr. Actin patches are repolarized in cells overexpressing the MID2 or SLG1 suppressors. Arrowheads point to the cortical actin patches and polarized rings, closed arrows point to the cable arrays, and open arrows to the unusual large actin clumps. (B) Transcription of MID2, SLG1, ACT1, or TUB1 remains largely unaffected with inactivation of STH1. Total RNA from wild-type (BLY76) and sth1-3ts (BLY49) cells grown at 30° for 8 hr following a temperature shift to 37° was fractionated and analyzed by Northern blot analysis. Experimental mRNA levels were normalized to ethidium bromide-stained rRNA and compared to wild-type RNA levels at 30° (arbitrarily set to 1.0).

We next investigated whether the high-copy suppressors could correct the delocalization of the cortical actin cytoskeleton in the sth1-3ts cells at 37°. In mutants carrying MID2 or SLG1, we detected polarized actin rings at the bud necks of large-budded cells and polarized actin patches and cable arrays in small- or medium-sized buds (Fig 6A). In addition, the unusual actin clumps characteristic of the mutants were seen only rarely. High-copy PKC1 also partially corrected the depolarized actin patches in sth1-3ts cells, although less effectively than did MID2 or SLG1 (data not shown).

One explanation for the ability of MID2 or SLG1 to suppress the ts lethality and/or the phenotypes of sth1-3ts is that transcription of these cell wall sensor genes requires STH1 function. However, we found that RNA levels of MID2 in sth1-3ts cells grown at 37° were comparable to those in wild-type cells shifted to 37°, and levels of SLG1 were, on average, only twofold lower (Fig 6B; data not shown). Alternatively, expression of genes that encode components of the cytoskeleton could be affected by loss of STH1 function. Again, transcription of ACT1, the sole actin gene in yeast, and TUB1, an -tubulin gene, was largely unaffected (Fig 6B). Thus, RSC appears to only mildly affect transcription of the genes that act in the alternative PKC1 effector pathway or the downstream cytoskeletal target genes.

MID2 or PKC1 partially suppresses the sth1 defects in mating projection formation:
Remodeling of the actin cytoskeleton is required for polarized cell growth in mitotically growing cells and projection formation during mating. The known involvement of Pkc1p signaling in actin cytoskeletal rearrangements prompted us to investigate pheromone-induced projection formation in the sth1-3ts cells (ERREDE et al. 1995 ; ZARZOV et al. 1996 ; BUEHRER and ERREDE 1997 ). Following a 3-hr exposure to -factor, 87% of sth1-3ts cells carrying wild-type STH1 formed mating projections while only 5% were found in S or G2/M cell cycle stages, consistent with results in wild-type cells. In contrast, 34% of -factor-treated sth1-3ts mutants carrying vector alone were still found in S or G2/M cell cycle stages and were therefore defective in responding to pheromone-induced late G1 arrest; untreated wild-type and mutant cells showed similar cellular distributions (Table 4). Significantly, 92% (87/87 + 8) of the unbudded sth1-3ts cells carrying STH1 formed mating projections or "shmoos," compared to only 29% (19/19 + 47) of the sth1-3ts cells carrying vector. (The percentage of shmoo formation is expressed as the fraction of cells forming shmoos in the unbudded population.) Moreover, compared to mutants expressing STH1, sth1-3ts cells carrying vector alone have a higher proportion of cells in G1 (47 vs. 8%) and S G2/M (34 vs. 5%), suggesting that the mutants are defective either in the remodeling of the actin cytoskeleton required for shmoo formation or in the cortical patch-dependent endocytosis critical for mating signal transduction.

The suppression of the actin cytoskeletal defects in budding sth1-3ts cells by Mid2p, Slg1p, or Pkc1p and the known involvement of these proteins in polarized growth for mating prompted us to test whether any of these proteins could rescue the mutant insensitivities to pheromone. We tested PKC1 and MID2 and found that either partially restored the response to mating pheromone in sth1-3ts mutants: 49% of mutants carrying PKC1 and 57% of mutants carrying MID2 formed shmoos. Additionally, fewer of these cells accumulated in either G1 or S G2/M.

The sth1 mutant sensitivity to the microtubule depolymerizing agent TBZ is corrected by high-copy suppressors:
Although a functional connection between PKC1 and the actin cytoskeleton is more firmly established, several reports indicate that PKC1 signaling is also functionally linked to the microtubule cytoskeleton (MANNING et al. 1997 ; KHALFAN et al. 2000 ). The tie between RSC and actin via the PKC1 pathway prompted us to test the role of RSC in microtubule function. A simple way to address this is to test the tolerance of sth1-3ts cells to disruption of microtubule structure. We found that mutant cells were hypersensitive to the microtubule-depolymerizing agent thiabendazole (TBZ) at 25°. The sth1 mutants shifted to the semipermissive temperature (35°) were also sensitive to TBZ (Fig 7). Interestingly, like suppression of the ts and cell cycle phenotypes, PKC1 and its upstream activators, MID2 and SLG1, also suppressed the TBZ growth inhibition of sth1-3ts mutants, but only at 35°. Downstream PKC1-MAP kinase module effectors failed to suppress the TBZ sensitivity, suggesting that the link to microtubule function is also via the alternative PKC1 pathway. Suppression at 35° but not 25° supports the notion that this alternative PKC1 pathway is more active at higher temperature.

View larger version (70K): In this window In a new window Download PPT slide   Figure 7. MID2, SLG1, or PKC1 can suppress the hypersensitivity of sth1-3ts to the microtubule-depolymerizing drug TBZ. Midlog phase sth1-3ts (BLY49) cells, carrying pBL50 (STH1), YEp24 (vector), pMID2 (MID2), pSLG1 (SLG1), pPKC1 (PKC1), or pMKK1 (MKK1) were 10-fold serially diluted, spotted on SC-Ura plates or SC-Ura plates containing 60 µg/ml TBZ, and incubated at 25° or 35° for 3–4 days.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Here, we have uncovered a link between Sth1p, the essential Snf2p-related ATPase component of RSC, and the Pkc1p-dependent signaling pathway. Our genetic analysis indicates that sth1 interacts specifically with an alternative PKC1 pathway that shares upstream activators with the PKC1-MAP kinase pathway. Moreover, we propose a model that this alternative pathway regulates the organization of the cellular cytoskeleton since this pathway rescues the sth1-3ts mutant defects in cortical actin polarization and its sensitivity to TBZ. Thus, our data provide important new insight into the cellular pathways in which RSC function is required.

RSC functions through an alternative PKC1-dependent pathway:
In addition to the SLG1 and MID2 transmembrane stress sensors isolated in our screen, ROM2, RHO2, and PKC1 were also shown to suppress the sth1-3ts ts growth defects. In contrast, the Pkc1p MAP kinase downstream effectors could not suppress sth1-3ts. Previously, several studies suggested pathways that branch downstream of Pkc1p. For example, the mating-induced death (Mid) phenotype of mid2 and the ts phenotype of glc7-10, an allele of a type 1 protein phosphatase, can be suppressed only by overexpression of PKC1 or its upstream activators (KETELA et al. 1999 ; ANDREWS and STARK 2000 ). Likewise, the transcriptional repression of ribosome synthesis and a novel arrest of secretion response in secretion-defective cells require only upstream elements of the PKC1 pathway (LI et al. 2000 ; NANDURI and TARTAKOFF 2001 ). Interestingly, suppression of the sth1-3ts G2/M arrest, actin delocalization, and TBZ sensitivity also distinguished upstream from downstream PKC1-MAPK components. Furthermore, high-copy MID2 can no longer suppress the sth1-3ts ts phenotype when the PKC1 pathway function is compromised by deletion of SLG1, indicating that PKC1 function is required for sth1-3ts suppression. Thus, several of the sth1-3ts defects are suppressed only by PKC1 and its upstream activators, strongly suggesting that RSC functions through an alternative PKC1-mediated signaling pathway, although we cannot rule out the possibility that RSC partly signals through the downstream MAP kinase cascade.

RSC function is necessary for cytoskeletal organization:
Our analysis of sth1-3ts cells has revealed a requirement for RSC function in actin cytoskeletal rearrangements necessary for polarized growth. First, the actin cytoskeleton is delocalized in mitotically arresting sth1-3ts cells at 37°. Second, sth1-3ts cells are defective in forming mating projections in the presence of mating pheromone, even at 30°.

The delocalized cortical actin patches observed in mitotically growing sth1-3ts cells at 37° have also been seen in pfy1 and rho1-2 mutants (HELLIWELL et al. 1998 ; MARCOUX et al. 2000 ). PFY1 encodes profilin, a small actin-binding protein proposed to function downstream of Rho1p. In addition, the unusual large actin clumps characteristic of sth1-3ts cells grown at the restrictive temperature have also been observed in sla1 and in nhp6A nhp6B double mutants (HOLTZMAN et al. 1993 ; COSTIGAN et al. 1994 ). Sla1p participates in cortical actin cytoskeleton assembly, and Nhp6A and Nhp6B are redundant HMG1-like DNA-binding proteins that function downstream of the PKC1-MAP kinase pathway. The inappropriate cortical patch distribution in sth1-3ts cells resembles that in mutants that affect aspects of actin cytoskeletal dynamics, implicating RSC in related actin-dependent functions.

A key event in polarizing the actin cytoskeleton in both mitotic growth and shmoo formation is the localization of the Rho-GTPase Cdc42p to selected growth sites. sth1-3ts mutants grown at the permissive temperature are capable of normal polarized mitotic cell growth but limited pheromone-induced polarized growth, suggesting that only the cell polarity signaling pathway unique to pheromone induction is defective at permissive temperature. Defects in the recruitment of Cdc42p or its effector kinases therefore appear unlikely. In addition, the ability of the majority of sth1-3ts cells to arrest in G1 in the presence of mating pheromone suggests that Far1p is still capable of inhibiting Cdc28p activity, which allows cells to enter S phase. Thus, we infer that downstream events required for actin cytoskeletal rearrangements are defective in sth1-3ts mutants.

Several lines of evidence also link RSC to microtubule-dependent processes via PKC1-dependent pathways. In this study, we showed that sth1-3ts cells exhibited hypersensitivity to TBZ, a microtubule depolymerizing agent, and that this sensitivity was suppressed by upstream PKC1 activators, including MID2. Previously, MID2 was shown to suppress mutations in Kar3p and Cik1p, two interacting proteins that regulate spindle pole body (SPB) and microtubule function (MANNING et al. 1997 ; BARRETT et al. 2000 ). A PKC1-mediated pathway has recently been shown to interact with components of the SPB as well (KHALFAN et al. 2000 ). In addition, an activator of the Rom2p exchange factor, Tor2p, transmits signals to the actin cytoskeleton and has been implicated in regulating microtubule structure and function (CHOI et al. 2000 ). Furthermore, the G2/M arrest of two rsc mutants has been shown to engage the MAD1-dependent spindle-assembly checkpoint (TSUCHIYA et al. 1998 ; ANGUS-HILL et al. 2001 ), a pathway that can be activated by defects in microtubule structure and function. More importantly, both the actin delocalization and TBZ sensitivity of sth1-3ts can be rescued by PKC1 and its upstream activators. Taken together, these data strongly suggest a requirement for RSC in actin cytoskeleton organization and microtubule function via a PKC1-dependent signaling pathway, thus linking nuclear chromatin remodeling activities with intracellular signaling.

Similar to the functional connection between hSWI/SNF and PKC/ras signaling, here we have demonstrated a link between RSC and a PKC1-dependent pathway. Although Pkc1p regulates diverse cellular processes, we show that RSC participates specifically in an alternative PKC1 pathway. Recent studies indicate that this alternative pathway may regulate interorganellar signaling from the secretory path to the nucleus (NANDURI and TARTAKOFF 2001 ). Interestingly, in an independent genetic screen, we recovered mutations in a secretory factor as extragenic suppressors of sth1-3ts multiple times (J. DU and B. C. LAURENT, unpublished results). Furthermore, defects in the secretory pathway can result in transcriptional repression of the genes encoding ribosomal proteins (RPs; WARNER 1999 ), and recently, the expression profiles of RP genes in two rsc mutants were shown to be altered (ANGUS-HILL et al. 2001 ). Our data connect RSC function to the actin cytoskeleton via the alternative PKC1 pathway. It will be important to examine expression levels of candidate target genes, e.g., RP genes, cell wall integrity genes, and secretory pathway genes. The connection between the alternative PKC1 and interorganellar secretory pathways further extends our model and supports the idea that RSC function is required for regulating actin-dependent secretion through this alternative PKC1 pathway.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724-2203.

	ACKNOWLEDGMENTS

We thank Pierre-Alain Delley for providing plasmids, Mara Amoros for assistance with RNA analysis, and members of the Laurent laboratory for helpful comments on the manuscript. This work was supported by Public Health Service grant GM56700 from the National Institutes of Health to B.C.L.

Manuscript received January 2, 2002; Accepted for publication March 18, 2002.

	LITERATURE CITED

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