© 2009 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).In Saccharomyces cerevisiae, the Rho-type GTPase Cdc42 regulates polarized growth through its effectors, including the p21-activated kinases (PAKs) Ste20, Cla4, and Skm1. Previously, we demonstrated that Ste20 interacts with several proteins involved in sterol synthesis that are crucial for cell polarization. Under anaerobic conditions, sterols cannot be synthesized and need to be imported into cells. Here, we show that Ste20, Cla4, and Skm1 form a complex with Sut1, a transcriptional regulator that promotes sterol uptake. All three PAKs can translocate into the nucleus and down-regulate the expression of genes involved in sterol uptake, including the Sut1 targets AUS1 and DAN1 by a novel mechanism. Consistently, deletion of either STE20, CLA4, or SKM1 results in an increased sterol influx and PAK overexpression inhibits sterol uptake. For Ste20, we demonstrate that the down-regulation of gene expression requires nuclear localization and kinase activity of Ste20. Furthermore, the Ste20-mediated control of expression of sterol uptake genes depends on SUT1 but is independent of a mitogen-activated protein kinase signaling cascade. Together, these observations suggest that PAKs translocate into the nucleus, where they modulate expression of sterol uptake genes via Sut1, thereby controlling sterol homeostasis.Deutsche Forschungsgemeinschaft and the Swiss National Science Foundation

Höfken, Thomas

Jacquier, Nicolas

Just, Ursula

Lin, Meng

Schneiter, Roger

Unden, Heike

Molecular Biology of the Cell

English

PubMed

Meng Lin

Heike Unden

Nicolas Jacquier

Roger Schneiter

Ursula Just

Thomas Höfken

MUCC (Crossref)

The Cdc42 Effectors Ste20, Cla4, and Skm1 Down-Regulate the Expression of Genes Involved in Sterol Uptake by a Mitogen-activated Protein Kinase-independent Pathway

Unden, H

Jacquier, N

Schneiter, R

Just, U

Höfken, T

Brunel University Research Archive

A dominant truncation allele identiﬁes a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae.

A genomewide screen reveals a role of mitochondria in anaerobic uptake of sterols in yeast.

A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae.

A membrane binding domain in the ste5 scaffold synergizes with gbetagamma binding to control localization and signaling in pheromone response.

A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1. Implications for modulation of mating pheromone signaling.

A role for cell polarity proteins in mitotic exit.

A system of shuttle vectors and yeast host strains designed for efﬁcient manipulation of DNA in Saccharomyces cerevisiae.

A versatile toolbox for PCR-based tagging of yeast genes: new ﬂuorescent proteins, more markers and promoter substitution cassettes.

Additional modules for versatile and economical PCR-based gene deletion and modiﬁcation in Saccharomyces cerevisiae.

An improved GFP cloning cassette designed for prokaryotic transcriptional fusions.

Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p.

Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20.

Cdc42-the centre of polarity.

Central roles of small GTPases in the development of cell polarity in yeast and beyond.

Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase.

Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p.

Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators.

Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast.

Control of Ltel localization by cell polarity determinants and Cdc14.

Coupling morphogenesis to mitotic entry.

Cytoplasmic localization of sterol transcription factors

Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth.

Elements of the yeast pheromone response pathway required for ﬁlamentous growth of diploids.

Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.

Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

Homologues of oxysterol-binding proteins affect Cdc42p- and Rho1p-mediated cell polarization in Saccharomyces cerevisiae.

Identiﬁcation of a UPC2 homolog in Saccharomyces cerevisiae and its involvement in aerobic sterol uptake.

Identiﬁcation of novel membranebinding domains in multiple yeast Cdc42 effectors.

Interaction of a G-protein beta-subunit with a conserved sequence in Ste20/PAK family protein kinases.

Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae.

Isolation and characterization of the Saccharomyces cerevisiae SUT1 gene involved in sterol uptake.

kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S.

Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae.

PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae.

Physiological implications of sterol biosynthesis in yeast.

Probing the molecular environment of membrane proteins in vivo.

protein kinase in yeast.

Proteins involved in sterol synthesis interact with Ste20 and regulate cell polarity.

regulate polarized growth in yeast.

Regulation of Sterol Uptake by PAKs

Rho GTPases: biochemistry and biology.

Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK,

Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae.

Septins, under Cla4p regulation, and the chitin ring are required for neck integrity in budding yeast.

Split ubiquitin as a sensor of protein interactions in vivo.

Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast.

Sterol-rich plasma membrane domains in fungi.

SUC1 and SUC 2, two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker’s yeast and identiﬁcation of the histidine-tagged protein.

SUT1 is a putative Zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells.

SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae.

synergistic action is sufﬁcient for this process.

The genetics of Pak.

The identiﬁcation of Pcl1-interacting proteins that genetically interact with Cla4 may indicate a link between G1 progression and mitotic exit.

The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate.

The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components.

Transcriptional proﬁling identiﬁes two members of the ATP-binding cassette transporter superfamily required for sterol uptake in yeast.

Where sterols are required for endocytosis.

Yeast mutants deﬁcient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions.

The Cdc42 effectors Ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway

In Saccharomyces cerevisiae, the Rho-type GTPase Cdc42 regulates polarized growth through its effectors, including the p21-activated kinases (PAKs) Ste20, Cla4, and Skm1. Previously, we demonstrated that Ste20 interacts with several proteins involved in sterol synthesis that are crucial for cell polarization. Under anaerobic conditions, sterols cannot be synthesized and need to be imported into cells. Here, we show that Ste20, Cla4, and Skm1 form a complex with Sut1, a transcriptional regulator that promotes sterol uptake. All three PAKs can translocate into the nucleus and down-regulate the expression of genes involved in sterol uptake, including the Sut1 targets AUS1 and DAN1 by a novel mechanism. Consistently, deletion of either STE20, CLA4, or SKM1 results in an increased sterol influx and PAK overexpression inhibits sterol uptake. For Ste20, we demonstrate that the down-regulation of gene expression requires nuclear localization and kinase activity of Ste20. Furthermore, the Ste20-mediated control of expression of sterol uptake genes depends on SUT1 but is independent of a mitogen-activated protein kinase signaling cascade. Together, these observations suggest that PAKs translocate into the nucleus, where they modulate expression of sterol uptake genes via Sut1, thereby controlling sterol homeostasis

Serveur académique lausannois

The Cdc42 effectors Ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway.

RERO DOC Digital Library

Supplemental figure legends  Figure S1. Overexpression of SUT1 under aerobic conditions induces expression of AUS1 and DAN1. Wild type cells carried either SUT1 under control of the strong PMA1 promoter on a multi copy plasmid (pNF1) or the corresponding empty vector (pNEV-N). In addition, cells harbored a plasmid on which the lacZ was fused to the promoter region of AUS1, DAN1 and PDR11, respectively. -galactosidase activity of the indicated strains was determined in at least 6 independent experiments.  Figure S2. Inhibition of sterol uptake by Ste20 is independent of the Ste20 NLS. STE20 and STE20NLS, respectively, were integrated into ste20 hem1 cells. Sterol uptake was measured as described in Figure 11A. Data are normalized to free sterols and steryl ester levels of STE20 cells after 24 hours of uptake.  1 Table S1. Yeast strains used in this study   Name   Genotype   Source or reference   This study CTY34 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 His3MX6-GAL1-3HA-YCK1 This study HUY28 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 This study HUY31 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1 This study HUY35 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 KanMX6-GAL1-3HA-STE20 hem1∆::His3MX6 This study HUY37 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 KanMX6-GAL1-3HA-SKM1 This study HUY42 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1 sut1∆::KanMX6 This study HUY43 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 ste11∆::hphNT1 This study HUY44 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 ste11∆::klTRP1 KanMX6-GAL1-3HA-STE20 This study HUY45 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 ste11∆::His3MX6 This study HUY47 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 ste11∆::hphNT1 KanMX6-GAL1-3HA-STE20 This study HUY50 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 His3MX6-GAL1-3HA-CLA4 This study HUY52 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 KanMX6-GAL1-3HA-CLA4 This study HUY55 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1 STE20K649A -URA3 This study MLY48 MATa his3::hisG leu2::hisG trp1::hisG ura3-52 ste20∆::KanMX6 HIS3-STE20272-288  This study MLY49 MATa his3::hisG leu2::hisG trp1::hisG ura3-52 ste20∆::KanMX6 HIS3-STE20 This study MLY107 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 His3MX6-GAL1-3HA-SKM1 This study MLY109 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 skm1∆::His3MX6 This study MLY113 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 skm1∆::klTRP1 His3MX6-GAL1-GFP-SKM1-LEU2 This study MLY114 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 His3MX6-GAL1-GFP-SKM1213-230 -LEU2 skm1∆::klTRP1 This study MLY180 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 ste20∆::KanMX6 STE20-His3 This study MLY181 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 ste20∆::KanMX6 STE20272-288-His3 This study MLY185 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 His3MX6-GAL1-3HA-SKM1 KanMX6-GAL1-3HA-CLA4 This study MLY186 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::hphNT1 cla4∆::klTRP1 This study MLY187 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 skm1∆::hphNT1 hem1∆::klTRP1 This study MLY197 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 His3MX6-GAL1-GFP-SKM1K213A/R214A-LEU2 skm1∆::klTRP1 This study MLY198 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 skm1∆::klTRP1 His3MX6-GAL1-GFP-SKM1R227A/K228A/K230A-LEU2 This study MLY199 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 skm1∆::klTRP1 SKM1-URA3 This study MLY200 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 skm1∆::klTRP1 SKM1NLS-URA3 This study MLY201 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 skm1∆::hphNT1 SKM1-URA3 This study MLY202 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 skm1∆::hphNT1 SKM1NLS-URA3 This study PPY966 MATa his3::hisG leu2::hisG trp1::hisG ura3-52 Tiedje et al. (2007) SHY41 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 sut1∆::His3MX6 KanMX6-GAL1-3HA-STE20 This study SHY43 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 sut1∆::His3MX6 ste20∆::klTRP1 This study SHY44 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 sut1∆::klTRP1  KanMX6-GAL1-3HA-STE20 This study SHY50 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 sut1∆::His3MX6 This study SHY70 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 sut1∆::hphNT1  KanMX6-GAL1-3HA-SKM1 This study SHY71 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 sut1∆: :hphNT1 His3MX6-GAL1-3HA-SKM1 This study SHY72 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 sut1∆::hphNT1 hem1∆::klTRP1  KanMX6-GAL1-3HA-CLA4 This study SHY73 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 ste20∆::klTRP1 STE20-URA3 This study SHY74 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1 STE20-URA3 This study SHY75 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 sut1∆::klTRP1 His3MX6-GAL1-3HA-CLA4 This study SHY79 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 KanMX6-GAL1-3HA-STE20-URA3 ste20∆::klTRP1 This study SHY80 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1  KanMX6-GAL1-3HA-STE20-URA3 This study THY310 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 ste20∆::klTRP1 Tiedje et al. (2007) THY547 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 sut1∆::His3MX6 This study THY566 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 KanMX6-GAL1-3HA-STE20 This study THY608 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 cla4∆::klTRP1 This study THY696 MATa his3::hisG leu2::hisG trp1::hisG ura3-52 ste20∆::KanMX6 This study THY725 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 ste20∆::KanMX6 This study THY732 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 His3MX6-GAL1-3HA-YCK1 This study THY734 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 skm1∆::KanMX6 This study THY737 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 cla4∆::KanMX6 This study THY738 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::klTRP1 KanMX6-GAL1-3HA-GIC2 This study THY741 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 KanMX6-GAL1-3HA-GIC2 This study THY742 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1 STE20272-284-URA3 This study THY743 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 ste20∆::klTRP1 STE20272-284-URA3 This study THY747 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1  KanMX6-GAL1-3HA-STE20K649A-URA3 This study THY748 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 hem1∆::His3MX6 ste20∆::klTRP1  KanMX6-GAL1-3HA-STE20272-284 -URA3 This study THY751 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 KanMX6-GAL1-3HA-STE20K649A-URA3  ste20∆::klTRP1 This study THY752 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 KanMX6-GAL1-3HA-STE20272-284 -URA3  ste20∆::klTRP1 This study THY754 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 ste20∆::klTRP1 STE20K649A-URA3 This study YPH499 MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1 Sikorski and Hieter (1989) YRS1707 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 Reiner et al. (2006) YRS1962 MAT his3∆1 leu2∆0 ura3∆0 lys2∆0 hem1∆::LEU2 aus1∆::KanMX4 pdr11∆::KanMX4 Reiner et al. (2006)       Table S2. Plasmids used in this study   Name   Genotype   Source or reference    pCT2 pRS313 carrying pMET25-RDI1-CUbiquitin-RURA3 Tiedje et al. (2007) pCT18 pET28c carrying SUT1 This study pCT54 pET28c carrying SEC6 This study pHU31 YEp367 carrying pAUS1 This study pHU32 YEp367 carrying pPDR11 This study pKA86 pRS316 carrying pGAL1-STE20-3HA Tiedje et al. (2007) pML32 pRS313 carrying STE20272-288 This study pML33 pRS313 carrying STE20 This study pML34 pPP1843 carrying STE20272-400 This study pML40 pPP1843 carrying STE20272-288** This study pML41 pPP1843 carrying STE20285-400 This study pML42 pPP2022 carrying STE20 This study pML47 pPP1843 carrying STE20272-600 This study pML49 pPP1843 carrying STE20272-288* This study pML55 pPP2022 carrying STE20272-284 This study pML56 pPP2022 carrying STE20272-288 This study pML78 pPP1843 carrying CLA4 This study pML88 pPP1843 carrying CLA4181-400 This study pML89 pPP1843 carrying CLA4181-550 This study pML90 pPP1843 carrying CLA4400-550 This study pML93 pPP1843 carrying CLA4450-550 This study pML94 pPP1843 carrying SKM1201-230 This study pMR216 YEp367 carrying pDAN1 Regnacq et al. (2001) pNEV-N 2m, URA3, pPMA1 Sauer and Stolz (1994) pNF1 pNEV-N carrying SUT1 Ness et al. (2001) pPP1843 pRS426 carrying pGAL1-GST-GFP Winters et al. (2005) pPP2022 pRS316 carrying pGAL1-GST-GFP Peter Pryciak pTH197 pRS313 carrying pMET25-STE20-CUbiquitin-RURA3 Tiedje et al. (2007) pTH283 pGEX-5X-1 carrying SUT1 This study pTH345 pADNX carrying pADH1-NUbiquitin-UBC6 Tiedje et al. (2007) pTH348 pADNX carrying pADH1-NUbiquitin-SUT1 This study YEp367 2m, LEU2, lacZ Myers et al. (1986)     *, fusion with the peptide SGAGAGAGAGAIL **, fusion with the peptide LGYFLFFEGGPGTQFAL  

The Cdc42 effectors ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway

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The Cdc42 effectors Ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway

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