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

Abstract

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. INTRODUCTIONThe small Rho GTPase Cdc42 plays a central role in the regulation of cellular polarity in eukaryotic cells (EtienneManneville, 2004;Jaffe and Hall, 2005). In the budding yeast Saccharomyces cerevisiae, Cdc42 regulates different types of polarized growth during various phases of its life cycle, including budding during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon nutrient limitation (Park and Bi, 2007). Cdc42 promotes polarized growth through multiple pathways, including polarization of the actin cytoskeleton and directed vesicle trafficking. Among the Cdc42 effectors that trigger these pathways are Ste20, Cla4 and Skm1, members of the p21-activated kinase (PAK) family of serine/threonine protein kinases (Hofmann et al., 2004;Park and Bi, 2007).Cdc42 recruits Ste20 and Cla4 from the cytoplasm and activates them at the plasma membrane at sites of polarized growth. Therefore, Ste20 and Cla4 are enriched at tips of buds and mating projections (Peter et al., 1996;Leberer et al., 1997;Holly and Blumer, 1999). The recruitment of Ste20 and Cla4 to these sites is governed by protein-protein as well as protein-lipid interactions. PAKs carry a conserved Cdc42/ Rac-interactive binding (CRIB) domain that mediates binding to Cdc42 and regulates their activity (Cvrckova et al., 1995;Peter et al., 1996;Leberer et al., 1997;Lamson et al., 2002;Ash et al., 2003). Ste20 and Cla4 also bind to Bem1, a scaffold protein that brings Cdc42, its activator Cdc24 and either Ste20 or Cla4, into proximity (Leeuw et al., 1998;Gulli et al., 2000;Bose et al., 2001;Yamaguchi et al., 2007). In addition, phosphoinositide-binding domains promote the association with membrane lip...

Regulation of Vacuolar H + -ATPase Activity by the Cdc42 Effector Ste20 in Saccharomyces cerevisiae

“…Ste20 has previously been known to localize to the plasma membrane at sites of polarized growth in the cytoplasm and the nucleus (1,25,28,43). Here, we show that Ste20 is also enriched in vacuolar membranes.…”

“…Ste20 promotes polarized growth, exit from mitosis, and cell death (1,16,53). It also regulates vacuolar inheritance, mRNA decay, and the synthesis and uptake of sterols (2,28,29,68). Furthermore, Ste20 activates distinct mitogen-activated protein kinase (MAPK) cascades that control filamentous growth, mating, and osmotic-stress responses (26,30,38,44,45,49).…”

The Zinc Cluster Protein Sut1 Contributes to Filamentation in Saccharomyces cerevisiae

“…This regulation requires an intact nuclear localization signal of Ste20, because Sut1 localizes exclusively to the nucleus (1, 7). In contrast, the nuclear localization signal of Ste20 is not required for its role in filamentation (7). This suggests that Ste20 does not play an important direct role in the regulation of the Sut1 protein during filamentation.…”

“…Instead, Sut1 could induce the expression of these genes by relief from repression by the general corepressor Cyc8-Tup1. Sut1 activity is regulated by the Cdc42 effectors Ste20, Cla4, and Skm1 (7). These proteins can form a complex with Sut1, and they can translocate into the nucleus, where they control the expression of genes whose products are involved in sterol uptake.…”

Mechanisms of sterol uptake and transport in yeast

“…For example, Rho-dependent polarized vesicular transport requires Osh protein function [57] and vesicular transport through the Golgi is affected by Osh4/Kes1 [58]. Effector kinases of the Rho-type GTPase Cdc42, Ste20 and Cla4, which are members of the p21-activated kinases (PAK) family, interact physically and genetically with sterol biosynthetic components and downregulate sterol levels under aerobic conditions as well as sterol uptake under anaerobic conditions, indicating a tight coordination of sterol levels with cell polarization [59][60][61]. Because bulk sterol transport from the ER to the plasma membrane is reduced rather than inhibited when Osh-function is depleted, it has been questioned whether Osh proteins have a direct role in bulk transport of sterols in vivo or if they have a more regulatory role on sterol transport or modulate signal transduction [38,62,63].…”