The Putative Lipid Transporter, Arv1, Is Required for ... - Genetics

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Nov 17, 2010 - ABSTRACT. Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projec- tions (shmoos), which ...
Copyright Ó 2011 by the Genetics Society of America DOI: 10.1534/genetics.110.120725

The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae Michelle L. Villasmil,*,† Alison Ansbach† and Joseph T. Nickels, Jr.*,†,1 *Pharmacogenomics Division, Medical Diagnostics Laboratories, Hamilton, New Jersey 08690 and †Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102 Manuscript received October 18, 2010 Accepted for publication November 17, 2010 ABSTRACT Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P2 and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5Q59L, which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P2 polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking.

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HE budding yeast Saccharomyces cerevisiae is viable as either a haploid or diploid cell. There are two haploid cell types, MATa and MATa (Mitchell 1994). MATa cells secrete a-factor pheromone and express the a-factor receptor, Ste2, while MATa cells secrete a-factor and express the a-factor receptor, Ste3 (Burkholder and Hartwell 1985; Nakayama et al. 1985). Ste2 and Ste3 are both seven-transmembrane proteins that activate the pheromone response pathway upon the binding of their cognate pheromone ligand (Cartwright and Tipper 1991). Ligand binding activates the receptor-bound G protein heterotrimer Gpa1-Ste4-Ste18, whereby the Gbg subunit (Ste4Ste18) dissociates from the Ga subunit (Gpa1) and subsequently transmits and amplifies the mating signal through effector pathways, which include a mitogenactivated protein (MAP) kinase cascade (Nakayama et al. 1988; Wang and Dohlman 2004). The Ste5 scaffolding protein recruits the MAP kinases Ste11, Ste7, and Fus3 in response to pheromone to initiate signaling. In turn, Ste5 is recruited to the plasma

1 Corresponding author: Pharmacogenomics Division, Medical Diagnostics Laboratories, 2439 Kuser Rd., Hamilton, NJ 08690. E-mail: [email protected]

Genetics 187: 455–465 (February 2011)

membrane by pheromone/receptor binding and subsequent interaction with the Gbg dimer Ste4-Ste18, but also interacts with phosphatidyinositol 4,5bisphosphate [PI(4,5)P2] in the plasma membrane through its pleckstrin homology (PH) domain (Winters et al. 2005; Garrenton et al. 2006, 2010). Activation of the pheromone response pathway results in G1 cell cycle arrest, mating-specific gene transcriptional induction, and changes in cytoskeletal structure, which allows for polarized cell growth and alterations in nuclear architecture, ultimately leading to cell fusion and formation of an a/a diploid (Wang and Dohlman 2004). In S. cerevisiae, pheromones stimulate the formation and growth of mating projections called shmoos. The site of polarization is determined by intrinsic and extrinsic signals. Pheromones act as an extrinsic signal to induce membrane polarity during mating (Madden and Snyder 1998). Yeast cells exhibit a chemotaxic response to pheromone of the opposite mating type, whereby they initiate shmoo formation toward the source of pheromone ( Jackson et al. 1991; Segall 1993). Once the site of shmoo formation has been established (Konopka et al. 1988; Chenevert et al. 1994), the GTPase Cdc42 signals reorganization of the cytoskeleton and other polarized components of the cell (Chang and Peter

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2003). A large protein complex, referred to as the polarisome, is also localized to the shmoo and is critical for proper shmoo site selection, formation, and growth (Bidlingmaier and Snyder 2004). The putative lipid transporter, Arv1, contains a region known as the Arv1 homology domain (AHD), which is conserved across several fungal and metazoan species, including humans. How the AHD functions within various Arv1-dependent events is not known. It is known that Arv1 function is essential at high temperatures and in yeast mutants unable to esterify sterols (Tinkelenberg et al. 2000). Moreover, arv1 cells harbor defects in sphingolipid metabolism (Swain et al. 2002), glycosylphosphatidylinositol (GPI) biosynthesis (Kajiwara et al. 2008), and may harbor defects in sterol trafficking (Tinkelenberg et al. 2000; Fei et al. 2008). It has been suggested that Arv1 plays a role in cholesterol trafficking from the ER to plasma membrane in mammalian cells (Tong et al. 2010). In the present work we show that arv1 yeast cells are mating defective. Our results demonstrate roles for Arv1 in pheromone-induced MAP kinase signaling and sterol microdomain localization. The defects in sterol trafficking may lead to defects in MAP kinase scaffold Ste5 localization to the plasma membrane by reducing PI(4,5)P2 polarization. Importantly, the AHD is required for all Arv1dependent mating functions. MATERIALS AND METHODS Strains, media, and miscellaneous microbial techniques: The yeast strains used in this study are derived from the W303 (MATa leu2-3, 112 trp1-1 ura3-1 his3-11, 15 can1-100) background. arv1Tkanr and bar1Tkanr strains were generated by a PCR-based knockout strategy using genomic DNA from arv1Tkanr and bar1Tkanr haploid strains, respectively, (Open Biosystems, Huntsville, AL) as a template. Yeast strains were grown in either YEPD (1% yeast extract, 2% bactopeptone, 2% glucose), or in synthetic minimal media containing 0.67% yeast nitrogen base (Difco, Sparks, MD) supplemented with the appropriate amino acids, adenine and uracil. Yeast transformation was performed using the procedure described by Ito et al. (1983). For routine propagation of plasmids, Escherichia coli XL1-blue cells were used and grown in LB medium supplemented with ampicillin (150 mg/ml). Bacterial transformations were carried out by electroporation methods. a-Factor was purchased from Sigma Chemicals (St. Louis, MO). All plasmids containing the AHD and Arv1 lacking AHD (DAHD) are under the control of the ARV1 promoter and are C-terminally tagged with 3HA in the vector pRS416 (Figure 1A). The following yeast strains were a gift from Peter Pryciak: MATa FUS1TFUS1-lacZTLEU2 (Strickfaden et al. 2007), MATa ste4TADE2 FUS1TFUS1-lacZTLEU2 (Pryciak and Huntress 1998), and MATa ste11TADE2 FUS1TFUS1-lacZT LEU2 (Pryciak and Huntress 1998). The following plasmids were a gift from Peter Pryciak: 2m HIS3 pGAL1-STE12 (Strickfaden and Pryciak 2008), CEN TRP1 pGAL1-STE11Cpr (Strickfaden et al. 2007), CEN TRP1 pGAL1-GST-Ste11DN (Moskow et al. 2000), CEN URA3 STE5-GFPx3 (Strickfaden et al. 2007), CEN URA3 pGAL1-STE5Q59L (Winters et al. 2005),

Figure 1.—Arv1 protein constructs. (A) A schematic of the various Arv1 protein constructs studied. Constructs are driven by the ARV1 promoter and are C-terminally tagged with 3HA in the vector pRS416. (B) Western analysis of cells expressing constructs described in A. Whole cell protein lysate was probed with anti-HA and anti-Pgk1. and CEN TRP1 pGAL1-STE5DN-CTM (Pryciak and Huntress 1998). The following plasmid was a gift from Jeremy Thorner: CEN LEU2 pGAL1-GST-GFP-PHPLCd (Winters et al. 2005; Garrenton et al. 2010). Protein extraction and immunoblotting: Protein was extracted using the NaOH/TCA cell lysis/protein extraction protocol as previously described (Garrenton et al. 2010). 3HA-tagged Arv1 constructs were detected using anti-HA (clone 16B12) from Covance (Princeton, NJ). Pgk1 was used as a loading control (Invitrogen). Qualitative limited mating plate assays: On day 1, cells to be assayed for mating were patched onto a YEPD plate and grown at 30° for 1 day to generate a master plate. In addition, cells of the opposite mating type were grown in liquid medium so that they reached exponential phase on day 2. On day 2, 1 3 106 of these exponentially grown cells were spread evenly across a YEPD plate and allowed to dry for several hours. The master plate was then replica plated onto the dried YEPD plate and incubated for 3 hr at 30°. Diploid progeny were then selected for using the appropriate minimal medium plate, and diploids were allowed to grow for 24 hr at 30°. Quantitative limited mating liquid assays: Cells were grown to exponential phase in synthetic complete or synthetic dropout medium. Cell density was determined at an optical density of 600 nm. A total of 1 3 107 cells of the mating control strain were mixed with 1 3 106 cells of the tester strain in YEPD. The cell mixture was transferred onto 0.45 mm MF membrane filters (Millipore, Billerica, MA), placed onto YEPD plates, and incubated for 3 hr at 30°. As a control for haploid cell growth, 1 3 106 cells of the tester strain were also transferred to a filter. After incubation, cells were transferred to microfuge tubes for 10-fold serial dilutions. One-hundred-microliter aliquots of cells from filters containing the mating control and tester strains were spread onto the appropriate synthetic minimal medium plates to select for diploid cells. Aliquots from filters that only contained the haploid tester strain were spread onto YEPD plates. Plates were incubated at 30° for 2 days and then colonies were counted. Mating efficiency was calculated as the number of diploid colonies divided by the number of haploid colonies. Determination of the rate of shmoo formation: MATa haploid cells were grown to 2.0 3 107 cells/ml in synthetic media. Cells were subsequently stimulated with 20 mg/ml

Arv1 Is Required for Yeast Mating

457 TABLE 1

Quantitative mating efficiencies of arv1 a and a haploids MATa (tester strain)

MATa (tester strain)

MATa WT arv1 MATa WT arv1 WT 24.1 6 2.4 1.8 6 1.1 WT 11.4 6 3.3 0.7 6 0.1 arv1 0.2 6 0.2 arv1 0.0 6 0.0 Mating efficiency is represented as the ratio of diploid colonies per haploid colonies counted (mean 6 SEM, n ¼ 3). WT, wild type.

Figure 2.—arv1 cells harbor bilateral mating defects. Cells to be tested for mating efficiency were patched onto YEPD plates and grown for 1 day (WT and arv1). A total of 106 cells of the opposite mating partner that were grown to exponential phase in liquid YEPD media were spread onto YEPD plates and allowed to dry (MATa WT; MATa WT; MATa arv1; MATa arv1). Mating was performed at 30° for 3 hr. Diploid progeny were selected by replica plating onto the appropriate media and allowed to grow for 1 day at 30°.

before they were visualized. Cells containing pGAL1-GST-GFPPHPLCd1 were grown in media containing 2% raffinose and induced with 2% galactose for 3 hr; 20 mg/ml of a-factor was added during the last hour of galactose induction. To examine sterol lipid distribution using filipin, cells were first grown to 2 3 107 cells/ml in synthetic medium. They were then fixed with 3.7% EM-grade formaldehyde for 10 min at 23° under constant mixing. Fixed cells were centrifuged and washed twice with distilled water. Washed cells were incubated with 100 mg/ml filipin complex (Sigma Chemicals) in the dark for 15 min at 23°. Filipin fluorescence was observed with UV optics using a Leica Digital Module R fluorescence microscope. Statistical analysis: Student’s t-tests were used to determine significance of data. All data were compared to results from wild-type cells. RESULTS

a-factor. Cell aliquots were taken from a-factor–treated cultures at 0, 1.5, 3, 4.5, and 7.5 hr, fixed with 3.7% electron microscopy (EM)-grade formaldehyde and washed twice with sterile water before visualizing shmoo formation using light microscopy and Nomarski optics. Three hundred cells were analyzed to determine the percentage of shmoo forming cells. Cell cycle analysis: For FACS experiments, MATa bar1 haploid cells were grown to 2 3 107 cells/ml in synthetic media. Cells were subsequently stimulated with 20 mg/ml a-factor and incubated at 30°. Cell aliquots were taken from a-factor–treated cultures at 0, 1.5, 3, 4.5, and 7.5 hr and fixed in 70% ethanol overnight at 20°. Cells were sonicated briefly to disrupt cell aggregates and incubated with 1 mg/ml RNAse A at 50° for 1 hr. Cells were incubated in 50 mg/ml propidium iodide (Sigma Chemicals) for 15 min at 4° and then washed before FACS analysis on a BD FACSCalibur. Data were analyzed using BD CellQuest software. b-Galactosidase assays: Activation of integrated FUS1-lacZ by galactose-inducible constructs was measured 4 hr after adding 2% galactose to cultures grown in 2% raffinose media. To measure effects of a-factor on FUS1-lacZ induction, cells were grown in 2% glucose media and induced with 10 mg/ml of a-factor for 2 hr. Total cell extracts were obtained and assayed for b-galactosidase activity using chlorophenol redb-d-galactopyranoside (CPRG) (Roche, Belvidere, NJ) as the substrate as described in the Clontech Yeast Protocols Handbook (Clontech Laboratories 2001). One b-gal unit is defined as the amount of b-galactosidase hydrolyzing 1 mmol of CPRG to chlorophenol red and d-galactose per minute per cell. Microscopy: Visualization of Ste5-GFPx3 and GST-GFPPHPLCd1 were done in live cells (without fixation). Images shown are representative of multiple experiments. Cells expressing Ste5-GFPx3 were treated with 20 mg/ml of a-factor for 1 hr

arv1 cells are defective in mating: During the course of isolating temperature-sensitive suppressors of arv1, we discovered that mutant haploids harbored defects in their ability to mate and form diploid progeny. We characterized this defect to gain a better understanding of the physiological functions of Arv1. We first used qualitative limited mating plate assays to determine whether the mating defect was mating-type specific (Chenevert et al. 1994). Both arv1 MATa and MATa cells harbored mating defects when mated to the opposite wild-type mating partner (Figure 2). Moreover, the mating defect was more severe when arv1 cells were mated to each other. Quantitative limited mating liquid assays showed that arv1 MATa cells had a 16-fold reduction in mating efficiency and arv1 MATa cells were 13-fold reduced when mated to wild-type cells (Table 1). Thus, arv1 cells harbor a unilateral mating defect (Berlin et al. 1991). In agreement with our qualitative plate assays, the low-level mating observed in our quantitative assay was further reduced when arv1 cells were mated to one another. We next wanted to know whether the conserved Arv1 homology domain was required for mating. Western analysis showed that 3HA-tagged Arv1 (Figure 1B, lane 1) and AHD (Figure 1B, lane 2) constructs were expressed equally; the DAHD construct was not detected. Because the DAHD partially rescues defects associated with ARV1 deletion (see below), we believe DAHD protein is present, but is localized to a fraction we are

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M. L. Villasmil, A. Ansbach and J. T. Nickels TABLE 2 Quantitative mating efficiencies of arv1 a and a haploids containing centromeric AHD-3HA, and DAHD-3HA plasmids MATa (tester strain) MATa Arv1 Mutanta

Arv1 17.1 6 2.8

AHD 15.8 6 2.4 11.2 6 3.1

MATa (tester strain) DAHD 6.5 6 1.6 1.7 6 0.2

MATa Arv1 Mutanta

Arv1 10.2 6 1.4

AHD 8.0 6 0.7 5.8 6 1.5

DAHD 2.5 6 1.3 0.3 6 0.0

arv1 a and a cells were transformed with plasmids containing full-length Arv1, AHD, and DAHD; the constructs are under the control of the ARV1 promoter and are C-terminally tagged with 3HA in the vector pRS416. Mating efficiency is represented as the ratio of diploid colonies per haploid colonies counted (mean 6 SEM, n ¼ 3). a Mutant is defined as the Arv1 mutant being tested (AHD or DAHD).

unable to recover using our protein isolation protocol. However, we point out the possibility that increased expression of the DAHD may have the ability to fully recover arv1-associated defects illustrated below. Quantitative mating assays revealed that MATa and MATa cells expressing only the AHD were mating competent when crossed to ARV1 cells (Table 2). Both MATa and MATa cells lacking the AHD (DAHD) had a three- to fourfold decrease, respectively, in mating efficiency when crossed to ARV1 cells. It must be pointed out that the decreased mating of DAHD cells was not as severe as arv1 matings (Table 1). On the basis of these results, we conclude that the AHD of Arv1 is required for wild-type mating. arv1 cells have defects in shmoo formation: To explore the reason for the mating defect, we first asked whether arv1 cells could form shmoos in the presence of pheromone. All strains were bar1, which sensitizes the cells to a-factor treatment; BAR1 encodes an aspartyl protease that is secreted by MATa cells and is responsible for the cleavage and inactivation of a-factor ( Jones 1984). arv1 bar1 MATa cells were incubated with 20 mg/ml of a-factor and the percentage of shmoos formed was determined over time using light microscopy. We observed that arv1 bar1 cells harbored a severe defect in shmoo formation when compared to the arv1 bar1 cells expressing full-length ARV1 (Figure 3). At 3 hr posta-factor treatment 65% of ARV1-expressing cells formed shmoos, as compared to only 3% in arv1 bar1 cells (Figure 3). When we expressed the AHD in arv1 bar1 cells, it restored shmoo formation to nearly ARV1 levels, while cells expressing DAHD had reduced shmoo formation (31% at 3 hr) (Figure 3). Thus arv1 bar1 cells harbor a defect in pheromone-induced polarized growth, which can be suppressed by expression of the AHD. arv1 cells cannot initiate pheromone-induced G1 cell cycle arrest: To further explore why arv1 cells are mating defective and unable to form shmoos, we assayed for their ability to initiate a-factor–induced G1 cell cycle arrest. We treated cells with a-factor and determined cell cycle status by performing FACS analysis on propidium iodide-stained cells. As shown in Figure 4, nearly all bar1 and arv1 bar1 cells expressing full-length ARV1 arrested after 3 hr, whereas arv1 bar1 cells were severely delayed in

their ability to arrest. During the 7.5-hr time course, arv1 bar1 cells did not enter complete G1 cell cycle arrest. Since expression of the AHD restored mating and shmoo formation in arv1 cells, we determined whether the AHD could remediate the pheromone-induced G1 cell cycle arrest phenotype. Cells expressing the AHD induced G1 cell cycle arrest by 3 hr, with the population of arrested cells being nearly identical to the wild-type population by 4.5 hr (Figure 4). On the other hand, cells expressing the DAHD were delayed in initiating cell cycle arrest and had fewer cells arrested in G1 phase at 3 hr when compared to bar1 cells (Figure 4). However, by 7.5 hr, more DAHD cells had entered G1 cell cycle arrest than arv1 bar1 cells. Thus, the AHD of Arv1 plays a major role in initiating pheromone-induced cell cycle arrest. The pheromone signaling pathway is defective in arv1 cells: In addition to stimulating Far1 to induce G1 cell cycle arrest, the MAP kinase pheromone response pathway activates the transcription factor Ste12, which binds to pheromone response elements within pheromone-regulated promoters and induces the tran-

Figure 3.—arv1 cells harbor defects in shmoo formation. A total of 2 3 107 cells/ml were incubated with 20 mg/ml of a-factor and cell aliquots were collected at 0, 1.5, 3, 4.5, and 7.5 hr. arv1 bar1 cells were transformed with the indicated Arv1 constructs which were C-terminally tagged with 3HA and on a pRS416 plasmid. Cells were collected at indicated time points and the numbers of shmoos/300 cells were counted for each sample. Arv1 full-length (solid circles), AHD (solid squares), DAHD (solid diamonds), empty vector (solid triangles) (mean 6 SEM, n ¼ 3, *P , 0.01, **P , 0.001).

Arv1 Is Required for Yeast Mating

Figure 4.—arv1 cells harbor defects in pheromoneinduced G1 cell cycle arrest. A total of 2 3 107 cells/ml of cells were treated with 20 mg/ml of a-factor for the indicated times. MATa bar1 and arv1bar1 cells were transformed with plasmids containing full-length Arv1, AHD, and DAHD, as indicated; the constructs are under the control of the ARV1 promoter and are C-terminally tagged with 3HA in vector pRS416. Data are shown as the quantification of FACS profiles measuring propidium iodide-stained DNA. G1 phase (light shading), S phase (solid), G2 phase, (dark shading).

scription of genes required for mating, including FUS1 (Kronstad et al. 1987; Van Arsdell et al. 1987; Dolan et al. 1989; Dolan and Fields 1990). Thus, we explored whether the cell cycle arrest phenotype of arv1 cells was due to defects in MAP kinase signaling. We used various strains harboring FUS1-lacZ integrated at the FUS1 locus as a readout for pathway activation. Cells were treated with a-factor and promoter activity was assayed after 2 hr. Under these conditions, wild-type cells had a relative FUS1 promoter activity that resulted in a steady state value of 44 b-gal units (Figure 5A). FUS1 promoter activity in arv1 cells was decreased by 50%. The expression of full-length Arv1 restored promoter activity to a value that was not statistically different from wild-type cells. Unexpectedly, we observed that FUS1 promoter activity in cells expressing the AHD was decreased when compared to wild-type cells (Figure 5A). This result correlates with the fact that arv1 cells ex-

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Figure 5.—arv1 cells harbor defects in pheromone response pathway signaling. (A) Pheromone signaling is defective in arv1 cells treated with a-factor. MATa FUS1TFUS1-lacZTLEU2 and arv1 FUS1TFUS1-lacZTLEU2 cells were transformed with plasmids containing full-length Arv1, AHD, and DAHD; the constructs are under the control of the ARV1 promoter and are C-terminally tagged with 3HA in vector pRS416. FUS1-lacZ induction was measured after 2 hr of a-factor treatment (mean 6 SEM, n ¼ 3, *P , 0.02, **P , 0.006). (B) Ste12 overexpression rescues signaling defect in arv1 cells. pGAL1STE12 was expressed in the strains described in A. FUS1-lacZ induction was measured 4 hr after galactose treatment (without a-factor) (mean 6 SEM, n ¼ 3).

pressing the AHD have a slight delay in initiating cell cycle arrest (Figure 4). arv1 cells expressing DAHD also showed a similar defect in FUS1 promoter activity. To further resolve whether MAP kinase signaling was defective in arv1 cells, we overexpressed Ste12 and examined FUS1 promoter activity. The overexpression of Ste12 has been shown to suppress the mating defect of ste mutants and increase transcription of pheromoneinducible genes (Dolan and Fields 1990). We observed that Ste12 overexpression rescued the signaling defect of arv1 cells and arv1 cells expressing the AHD or DAHD constructs (Figure 5B). Together, these data suggest that arv1 cells have a defect in pheromone-induced MAP kinase signaling that results in the inability to induce cell cycle arrest. Plasma membrane recruitment of the MAP kinase scaffold Ste5 suppresses the signaling defects of arv1 cells: Since arv1 cells have a weakened pheromoneinduced MAP kinase signaling response (Figure 5), we explored whether activating the pathway through constitutive Ste5 plasma membrane targeting had any effect on signaling in arv1 cells. Ste5 can be targeted artificially to the plasma membrane to activate the mating pathway in the absence of Gbg or pheromone (Pryciak

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M. L. Villasmil, A. Ansbach and J. T. Nickels Figure 6.—Ste5 recruitment to plasma membrane is altered in arv1 cells. (A) Pheromone signaling is rescued by overexpression of Ste5Q59L. pGAL1-Ste5Q59LGFP was expressed in strains described in Figure 5A. FUS1-lacZ induction was measured 4 hr after galactose treatment (without a-factor) (mean 6 SEM, n ¼ 3). (B) Expression of Ste5DN-CTM does not rescue signaling defect in arv1 or DAHD cells. pGAL1-Ste5DN-CTM-GFP was expressed in strains described in Figure 5A. FUS1-lacZ induction was measured 4 hr after galactose treatment (without a-factor) (mean 6 SEM, n ¼ 4, *P , 0.01). (C) Ste5-GFP was expressed in cells. Cells with polarized Ste5-GFP were counted after 1 hr of a-factor treatment (mean 6 SEM, n ¼ 4, *P , 0.001). (D) Cells with pGAL1-GSTGFP-PHPLCd were treated with galactose for 3 hr. a-Factor was added during the last 1 hr of galactose treatment. Cells with polarized PI(4,5)P2 were counted (mean 6 SEM, n ¼ 3, *P , 0.005, **P , 0.001).

and Huntress 1998; Winters et al. 2005). Plasma membrane-targeted Ste5 still requires Ste20 p21-activated kinase, the Cdc24 guanine exchange factor and Cdc42 (Pryciak and Huntress 1998; Winters et al. 2005). One mutant, Ste5Q59L, is targeted to the membrane because it harbors a more hydrophobic PM domain (Winters et al. 2005). The Ste5DN-CTM chimera, which lacks a portion of its amino terminus, is targeted to the membrane by the Snc2 transmembrane domain and it also activates MAP kinase signaling (Pryciak and Huntress 1998). We overexpressed these artificially targeted forms of Ste5 in cells and assayed FUS1-lacZ promoter activity. Ste5Q59L was able to activate MAP kinase signaling to a similar extent in all strains tested (Figure 6A), whereas Ste5DN-CTM required the presence of either full-length Arv1 or the AHD for full activation (Figure 6B). arv1 and DAHD cells expressing Ste5DN-CTM had diminished FUS1-lacZ activity (Figure 6B), whereas arv1 and DAHD cells expressing Ste5Q59L (Figure 6A), which binds more efficiently to the membrane, were signaling competent. These data suggest that Arv1 function, in particular AHD activity, may be required for efficient targeting of Ste5 to the plasma membrane in response to pheromone signaling. Thus, the mating defects of arv1 cells may result from mislocalization of Ste5. To more directly address the role of Arv1 in Ste5 recruitment to the plasma membrane, we treated cells expressing GFP-tagged Ste5 with a-factor for 1 hr and counted cells with polarized Ste5. Approximately 30% of wild-type cells had polarized Ste5-GFP, while only 16% of arv1 cells contained polarized Ste5-GFP (Figure 6C). Expression of Arv1, AHD, and DAHD in arv1 cells rescued the Ste5-GFP localization defect (Figure 6C).

These data suggest that Ste5 is not localized efficiently in arv1 cells during mating and that expression of the N-terminal or C-terminal segments of Arv1 recover the Ste5 localization defect. It has been shown that polarized PI(4,5)P2 in the plasma membrane interacts with the pleckstrin homology (PH) domain in Ste5 to cluster Ste5 (Winters et al. 2005; Garrenton et al. 2006, 2010) and that ergosterol promotes PI(4,5)P2 polarity ( Jin et al. 2008). To determine whether the sterol trafficking defects in arv1 cells contribute to defects in PI(4,5)P2 polarization in response to pheromone, we examined the localization of GST-GFP-PHPLCd after treating cells with a-factor. The PH domain of mammalian PLCd binds to PI(4,5)P2 with high specificity and affinity (Lemmon and Ferguson 2000; Hurley and Meyer 2001). A total of 41% of wildtype cells have polarized PI(4,5)P2 while only 1% of arv1 cells contain polarized PI(4,5)P2 after a-factor treatment (Figure 6D). Expression of full-length Arv1 rescues defects in PI(4,5)P2 polarization. Expression of the AHD partially rescues the PI(4,5)P2 polarity defect seen in arv1 cells (15%) while expression of DAHD does not (2.5%) (Figure 6D). Data are consistent with a model in which the sterol trafficking defects of arv1 cells disrupt PI(4,5)P2 polarization and, in turn, lead to diminished polarization of the MAP kinase scaffold Ste5 in response to pheromone. Expression of N-terminally truncated Ste11 partially suppresses the signaling defects of arv1 cells: In an attempt to bypass the need for pheromone-induced Ste5 membrane localization, we investigated whether expressing a catalytically active form of the MAP kinase kinase kinase Ste11 had any effect on the signaling defect of arv1 cells. Activation of Ste11 requires Ste20

Arv1 Is Required for Yeast Mating

Figure 7.—Activated Ste11 mutants do not rescue signaling defect in arv1 cells. (A) Expression of Ste11-Cpr does not rescue signaling defect in arv1, AHD, or DAHD cells. pGAL1-Ste11-Cpr was expressed in MATa ste11TADE2 FUS1T FUS1-lacZTLEU2 or MATa ste11TADE2 arv1TKANr FUS1T FUS1-lacZTLEU2 cells. FUS1-lacZ induction was measured 4 hr after galactose treatment (without a-factor) (mean 6 SEM, n ¼ 3, *P , 0.02, **P , 0.001). (B) Ste11DN expression does not rescue signaling defect in arv1 or DAHD cells. pGAL1-Ste11DN was expressed in strains described in Figure 5A. FUS1-lacZ induction was measured 4 hr after galactose treatment (without a-factor) (mean 6 SEM, n ¼ 4, *P , 0.01, **P , 0.001).

and Ste5 and results in amplification of the pheromone response through MAP kinase signaling (Pryciak and Huntress 1998; van Drogen et al. 2000; Lamson et al. 2006). We used two Ste11 mutants for our studies: Ste11Cpr (Strickfaden et al. 2007) and Ste11DN (Moskow et al. 2000; Strickfaden et al. 2007). Ste11-Cpr contains the membrane targeting sequence of Ras2. Expression of Ste11-Cpr causes constitutive activation of MAP kinase signaling (Winters et al. 2005). Ste11DN lacks the N-terminal regulatory domain that binds to and inhibits the C-terminal kinase domain (Wu et al. 1999; van Drogen et al. 2000). Ste11DN is considered a fully activated form of Ste11 and its expression also causes constitutive activation of MAP kinase signaling (Winters et al. 2005). It is under debate whether these mutants require Ste5 for full activation (Stevenson et al. 1992; Elion 1995). When we overexpressed Ste11-Cpr in arv1 cells, we found they still harbored a severe defect in MAP kinase signaling, retaining only 30% of the FUS1-lacZ promoter activity observed in wild-type cells (Figure 7A); arv1 cells treated with a-factor retain 55% FUS1-lacZ promoter activity (Figure 5A). Although promoter activity did increase in arv1 cells expressing the AHD or DAHD, the

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values were still decreased from that seen in wild-type cells (66 and 49%, respectively). Thus the overexpression of Ste11-Cpr only weakly suppressed the signaling defects of arv1 cells harboring the AHD or DAHD functional domains of Arv1. When we overexpressed Ste11DN in arv1 cells, it stimulated FUS1-lacZ promoter activity to a greater extent than Ste11-Cpr (67 vs. 30% of wild-type stimulation), but it remained reduced when compared to wildtype cells (Figure 7B). However, in contrast to Ste11-Cpr, overexpression of Ste11DN in arv1 cells expressing the AHD alone restored promoter activity to nearly a wildtype value (93%). Interestingly, expressing the DAHD in arv1 cells in the presence of Ste11DN had a dominant negative effect on promoter activity, as activity was only 50% of that seen in arv1 cells expressing Ste11DN. The data together demonstrate that the membrane targeted form of Ste11, Ste11-Cpr, only weakly suppresses the MAP kinase signaling defect of arv1 cells. We also demonstrate that the AHD alone is sufficient to fully activate MAP kinase signaling in the presence of Ste11DN, pointing to a critical role for this domain in pheromone signaling. Finally, our data suggest that Ste11-Cpr and Ste11DN act differently to activate pheromone-induced MAP kinase signaling. Overexpression of Gb Ste4 does not recover pheromone signaling defect of arv1 cells: As discussed above, the pheromone-activated Gbg Ste4-Ste18 heterodimer plays a role in recruiting Ste5 to the plasma membrane (Winters et al. 2005). If the pheromoneinduced MAP kinase signaling defect seen in arv1 cells arises from weakened pheromone-receptor signaling, rather than from poor trafficking of Ste5 to the plasma membrane, we would hypothesize that Ste4 (Gb) overexpression in arv1 cells would remediate the MAP kinase signaling defect (Figure 5A). As shown in Figure 8, Ste4 overexpression failed to rescue the MAP kinase signaling defects observed in arv1 and arv1 cells expressing the AHD or DAHD. The percentages of various unesterified sterol lipid microdomains are altered in arv1 cells: There has been controversy regarding the validity of filipin staining of unesterified sterols. Because we prefer to use this technique to examine sterol trafficking in response to pheromone, we attempted to resolve this issue. It has been demonstrated that filipin fluorescence is significantly diminished in cells unable to generate sterols; cells were fixed prior to filipin staining (Beh and Rine 2004). Another study used live cells; these cells were first stained with filipin and then subsequently stained with FM4-64. It was demonstrated that the vacuolar dye FM4-64 colocalized with filipin (Valdez-Taubas and Pelham 2003). Using the live cell staining protocol, we found that cell viability was severely compromised within 5 min of staining cells with filipin. Filipin belongs to the polyene class of antifungal drugs and acts by binding to membrane sterols and altering membrane perme-

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Figure 8.—Ste4 overexpression does not rescue arv1 signaling defect. pGAL1-Ste4-GFP was expressed in MATa ste4TADE2 FUS1TFUS1-lacZTLEU2 or MATa ste4 TADE2 arv1TKANr FUS1TFUS1-lacZTLEU2 cells. FUS1-lacZ induction was measured 4 hr after galactose treatment (without a-factor) (mean 6 SEM, n ¼ 4, *P , 0.02, **P , 0.01).

ability (Kawasaki et al. 1985). On the basis of our results, we believe that the observed filipin/FM4-64 colocalization is an artifact of membrane permeabilization and cell death by filipin, which allows for rapid uptake of FM4-64 at the site of membrane damage (ValdezTaubas and Pelham 2003). Beh and colleagues have used filipin staining to examine the cellular localization of unesterified sterol (Fei et al. 2008). The majority was found within lipid microdomains that were associated with the plasma membrane, with cytoplasmic punctate structures, and with membrane strands that may be associated with the ER. On the basis of the percentage distribution within these structures and the differences they observed between arv1 and wild-type cells, they hypothesized that Arv1 regulates the movement and localization of unesterified sterol in cells. Since others have also suggested a role for Arv1 in lipid trafficking (Tinkelenberg et al. 2000), we decided to examine the distribution of sterol within these various microdomains during mating using filipin to see whether arv1 cells could distribute sterol properly during conjugation. The lipid microdomain distribution we observed during vegetative growth in wild-type and arv1 cells grown in the absence of a-factor were similar to those previously reported (Fei et al. 2008). Thus, we treated cells with a-factor and determined the percentages of the various filipin staining patterns (Figure 9). We observed several trends emerging in treated bar1 cells. First, 76% of shmoo-containing cells localized a portion or all of their unesterified sterol to the shmoo tip [Table 3; polar, membrane strand (MS) polar, and cytoplasmic punctate (pun) polar]. Second, a large percentage (83%) of these cells also had sterol localized to cytoplasmic punctate or membrane strand structures (Table 3; MS polar and pun polar). These results indicate that the distribution/trafficking of sterol between these structures is dynamic and influenced by the initiation of mating and polarized shmoo formation.

Figure 9.—Fluorescence microscopic visualization of the lipid microdomains in cells during vegetative growth and a-factor treatment. The various lipid microdomains indicated in Table 3 are represented. Arrows indicate location of membrane strands.

Since arv1 bar1 cells are severely delayed in shmoo formation (Figure 3), we examined sterol localization at 7.5 hr post-a-factor treatment to see whether cells eventually distributed their sterol as wild-type cells did at 3 hr. We chose not to determine values for bar1 cells at 7.5 hr posttreatment, as the majority of cells contained more than one shmoo or a single highly elongated shmoo (Bidlingmaier and Snyder 2004). Importantly, we found that arv1 bar1 cells continued to have altered sterol distribution patterns. While 76% of bar1 cells containing shmoos polarized their sterol at 3 hr, only 38% of arv1 bar1 cells with shmoos were within the polarized categories at 7.5 hr (Table 3). Moreover, the arv1 bar1 cell population containing shmoos had a greater percentage of cells with membrane strandlocalized sterol (Table 3; MS) than the wild-type population (Table 3; MS). Thus, the sterol distribution within these dynamic structures during mating is greatly altered in arv1 bar1 cells. We then asked whether the expression of the AHD or DAHD in arv1 bar1 cells could restore normal sterol trafficking. After a 3 hr a-factor treatment, arv1 bar1 cells expressing the AHD localized sterols to the shmoo tip with a similar efficiency as wild-type cells (66 vs. 78%, data not shown), while only 35% of the DAHD cells had properly localized their sterol content (data not shown). Thus the AHD of Arv1 is sufficient to initiate and maintain pheromone-induced sterol trafficking during mating.

DISCUSSION

On the basis of our data, we believe that failure of arv1 cells to polarize PI(4,5)P2 in response to pheromone treatment results in diminished polarization of the MAP kinase scaffold protein Ste5; this leads to weakened MAP kinase signaling and decreased mating in arv1 cells. Defects in sterol microdomain trafficking and localization in response to pheromone treatment in arv1 cells may also play a role in the mating defect. Importantly, our studies show that the conserved AHD of Arv1 is sufficient for pheromone-induced G1 cell cycle arrest, shmoo formation, proper pheromone-induced sterol

Arv1 Is Required for Yeast Mating

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TABLE 3 Percentage distribution of filipin-stained lipid microdomain structures in a-factor–treated cells 1 a-Factor; shmoos Strains bar1 (3 hr) arv1bar1 (7.5 hr)

a

Rim %

Polar %

MS %

MS polar %

Pun polar %

nb %

15.3 38.2

12.6 30.0

9.1 23.6

39.4 8.2

23.6 0.0

254 110

Exponentially grown cells were treated with a-factor as indicated and then stained with filipin. a Lipid microdomain categories: MS, membrane strands; Pun, cytoplasmic punctate. b Number of cells counted.

trafficking, and subsequent conjugation and progeny formation. arv1 cells harbor defects in sphingolipid and GPI synthesis, and although their overall sterol content is normal, our data, as well as that of others, strongly suggest these lipids are trafficked improperly and thus are mislocalized (Tinkelenberg et al. 2000; Swain et al. 2002; Fei et al. 2008; Kajiwara et al. 2008). Membrane microdomains, also known as lipid rafts, are sphingolipidand sterol-rich regions of the plasma membrane, which are capable of mediating membrane sorting, cell adhesion, and signal transduction (Harder et al. 1998). Lipid rafts are capable of polarized protein recruitment that results in signal transduction (Simons and Ikonen 1997; Brown and London 1998; Simons and Toomre 2000). S. cerevisiae displays lipid-dependent cell polarity during cell budding and mating (Chant 1999). erg6 and lcb1-100 lcb3 mutants, which lack D(24)-sterol C-methyltransferase and serine palmitoyltransferase-long chain sphingoid base kinase, respectively, do not polarize Fus1 to the shmoo tip and have reduced mating efficiency (Bagnat and Simons 2002a). Fus1, a membrane protein that localizes to the shmoo tip, is required for cell fusion (Trueheart et al. 1987; Trueheart and Fink 1989). Moreover, lipid rafts preferentially localize to the shmoo tip during mating (Bagnat and Simons 2002a), suggesting an important role for these lipid microdomains in generating membrane polarization during conjugation. Our data further support this idea. There are many instances demonstrating roles for membrane lipids in signaling. Disrupting ergosterol or sphingolipid biosynthesis with mutations to erg2, erg3, and lcb1ts reduces a-factor–induced FUS1 transcription ( Jin et al. 2008). Moreover, erg3 mutants do not effectively recruit GFP-Ste5 to shmoo tips and it has been shown that overexpression of the membrane-targeted chimera Ste5DN-CTM in ergosterol-depleted cells partially rescues FUS1 transcription ( Jin et al. 2008). arv1 cells have a slight defect in the ER-to-Golgi transport of the GPI-anchored proteins Gas1 and Yps1, while there is no vacuolar trafficking defect in carboxypeptidase Y (CPY) (Kajiwara et al. 2008). Thus general vesicle-mediated trafficking is only slightly affected in arv1 cells and most likely does not contribute to the mating defects observed. In addition, the kinetics of

a-factor internalization in mutant cells is similar to that seen in wild type, indicating that pheromone receptors are localized properly and are functional (Kajiwara et al. 2008). Therefore, it is unlikely Ste5 mislocalization results from defects in general protein trafficking. In addition to having defects in sphingolipid and GPI syntheses, arv1 cells harbor defects in phospholipid biosynthesis and metabolism (Swain et al. 2002). Ste5 has an N-terminal amphipathic a-helix, which is important for nuclear localization and membrane binding (Winters et al. 2005). Ste5 also contains a PH domain, which is required for Ste5 recruitment to the plasma membrane and for its function. Importantly, the Ste5 PH domain binds to PI(4,5)P2 (Winters et al. 2005; Garrenton et al. 2006, 2010). Interestingly, it has been shown that erg6 mutants have less PI(4,5)P2 polarized to the shmoo tip, potentially influencing membrane recruitment of the PI(4,5)P2 binding proteins Ste5 and Far1 ( Jin et al. 2008). We have shown that arv1 cells have a PI(4,5)P2 polarization defect when treated with a-factor (Figure 6D). We believe that altered sterol distribution in polarized arv1 cells affects PI(4,5)P2 polarization, which in turn affects Ste5 recruitment. Ste5 is also tethered to the plasma membrane by interaction with activated Gbg and directly associates through a plasma membrane binding domain (Pryciak and Huntress 1998). Expression of the AHD partially recovers defects in PI(4,5)P2 polarization and fully recovers defects in Ste5 recruitment. These results suggest that expression of the AHD alone can remediate some of the lipid/sterol defects of arv1 cells. It is also of note that expression of the DAHD recovers defects in Ste5 recruitment but has no effect on defects in PI(4,5)P2 polarization. This suggests that in cells expressing the DAHD, Ste5 is able to associate with the plasma membrane by interacting through other membrane-associating domains. The AHD is highly conserved in many species, including yeasts, plants, and animals (Tinkelenberg et al. 2000). While only 61 residues long, expression of the AHD in arv1 cells rescues defects in pheromoneinduced G1 cell cycle arrest, shmoo formation, sterol trafficking, mating, and pheromone signaling when we express Ste5-CTM, Ste11DN, and to a lesser extent, Ste11-Cpr. We used the fluorescent polyene compound

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filipin to examine ergosterol trafficking and localization in fixed cells. By fixing the cells in formaldehyde, we effectively stop cellular activity and can evaluate ergosterol localization in static cells. Fixation permeabilizes cells, allowing for filipin staining of intracellular ergosterol. We believe that while filipin staining can be used in live cell experiments, it must be done quickly to avoid cell death and aberrant membrane localization of filipin; this method has been properly demonstrated (Bagnat and Simons 2002a). There is accumulating data supporting the role of lipid rafts in cell polarity in S. cerevisiae (Bagnat and Simons 2002a,b; Rajendran and Simons 2005). Yeast exhibit cell surface polarity during budding and mating (Harder et al. 1998). However, we point out that the existence and function of lipid rafts in S. cerevisiae is still the subject of debate (Wachtler and Balasubramanian 2006). In mammalian cells, lipid rafts are well characterized, and they play an important role in signal transduction and cell motility (Hoekstra et al. 2003). In general terms, they are critical for and have a fundamental role in the delivery of various cargos to different parts of the plasma membrane (Schuck and Simons 2004). For example, they target proteins and lipids to basolateral and apical membrane domains during cell polarity development of epithelial cells and regulate the transport of proteins to the axonal and somatodendritic domains in neurons (Hoekstra et al. 2003). Here we have demonstrated a role for Arv1 and the AHD in sterol trafficking and localization, in our case a critical function required for mating. The conservation of Arv1 among eukaryotes, including humans (Swain et al. 2002; Fores et al. 2006), points to the metazoan protein regulating lipid-dependent polarity. We thank Peter Pryciak and Jeremy Thorner for strains and plasmids. We thank Tom Edlind, Eishi Noguchi, Chris Burd, Elias Spiliotis, Martin Adelson, and Eli Mordechai for helpful discussions. This work was supported in part by National Institutes of Health grant HL67401. We also acknowledge and greatly appreciate the financial support of Medical Diagnostic Laboratories.

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Communicating editor: M. D. Rose