Cell Polarity in Yeast

Abstract

A conserved molecular machinery centered on the Cdc42 GTPase regulates cell polarity in diverse organisms. Here we review findings from budding and fission yeasts that reveal both a conserved core polarity circuit and several adaptations that each organism exploits to fulfill the needs of its lifestyle. The core circuit involves positive feedback by local activation of Cdc42 to generate a cluster of concentrated GTP-Cdc42 at the membrane. Species-specific pathways regulate the timing of polarization during the cell cycle, as well as the location and number of polarity sites.

Keywords: Cdc42; GAP; GEF; cell polarity; positive feedback; yeast.

Figure 1

Cdc42 regulators and effectors. Like other G proteins, Cdc42 exhibits slow intrinsic rates of GDP/GTP exchange and GTP hydrolysis, which are accelerated by GDP/GTP exchange factors (GEFs) or GTPase-activating proteins (GAPs). Cdc42 is prenylated at its C terminus, and much of the cell’s Cdc42 is found on the cytoplasmic face of cellular membranes, including the plasma membrane (Erickson et al. 1996, Richman et al. 2002, Ziman et al. 1993). A soluble pool of Cdc42 is also present and bound to GDIs (guanine nucleotide dissociation inhibitors) that can extract it from the membrane (Koch et al. 1997, Leonard & Cerione 1995, Leonard et al. 1992, Masuda et al. 1994, Tiedje et al. 2008). Effectors bind specifically to GTP-Cdc42, and not to GDP-Cdc42.

Figure 2

Polarity in Saccharomyces cerevisiae and Schizosaccharomyces pombe. (a) In S. cerevisiae, cells polarize Cdc42 in late G1 at a single site and grow a bud starting in S phase. Cdc42 becomes depolarized within the bud in G2 and concentrates (in the inactive form) at the mother-bud neck during cytokinesis. (b) In S. pombe, germinating spores break symmetry to polarize at a single site to generate a cylindrical cell. In rich media, cytokinesis and cell separation take a significant fraction of the cell cycle, and rapid G1 and S phases occur during this time, so cells are born in G2. Initially, cells polarize Cdc42 to the old end (opposite the cytokinesis site). After further growth, a second polarity site is established at the new end, and cells grow at both ends until mitosis, when Cdc42 relocates from the ends to the middle of the cells.

Figure 3

Positive feedback by local activation of Cdc42. (a) GTP-Cdc42 can bind effectors [p21-activated kinases (PAKs)] that in turn bind the scaffold Bem1, which binds the GDP/GTP exchange factor (GEF) Cdc24. (b) The PAK-Bem1-GEF complex allows one GTP-Cdc42 to promote activation of neighboring GDP-Cdc42 in a positive feedback loop. (c) Over time, positive feedback promotes local accumulation of GTP-Cdc42 and PAK-Bem1-GEF complexes at the membrane, depleting cytoplasmic pools of these components.

Figure 4

Effect of actin-mediated vesicle delivery on Cdc42. (a) In Saccharomyces cerevisiae, actin cables become oriented toward the polarity site by formins, and secretory vesicles are delivered to the polarity site by type V myosin. Exocyst complexes bind Cdc42 and promote vesicle fusion (exocytosis). (b) Vesicles lack most polarity factors, so vesicle fusion transiently dilutes polarity factor concentration, and off-center fusion of vesicles can lead to displacement of the centroid of the polarity site away from the vesicle fusion site.

Figure 5

Effect of microtubules and cell shape on Cdc42 in Schizosaccharomyces pombe. (a) Microtubule bundles align longitudinally within the cell, with plus ends pointing toward cell tips and minus ends in the cell middle. (b) Tip factors (Tea1, Tea4) are delivered to cell ends and bring binding partners, including protein phosphatase 1 (PP1), which promotes localization of the GDP/GTP exchange factor (GEF) Gef1 and exclusion of the GTPase-activating protein (GAP) Rga4 from cell ends. The spatial separation of GEF and GAP promotes Cdc42 activation at cell ends and inactivation at cell sides. (c) Cdc42 is further concentrated by the action of a positive feedback loop similar to that in Saccharomyces cerevisiae, with Scd1 = Cdc24 and Scd2 = Bem1.

Figure 6

Competition between polarity sites in Saccharomyces cerevisiae. (a) Cells often initially make >1 cluster of Cdc42 and other polarity factors but then eliminate all but one cluster, which goes on to form the bud. (b) Computational models of local activation initiated with two peaks of Cdc42 concentration show competition such that one peak grows and the other shrinks. Polarity factors exchange dynamically between membrane clusters and the shared cytoplasm, and in the computational model this exchange allows a larger peak to outcompete a smaller peak. (c) In cells genetically manipulated to slow the exchange of polarity factors between membrane and cytoplasm, competition slows, and cells can make >1 bud simultaneously. Adapted from Wu et al. (2015).

Figure 7

Size of polarity sites: GTPase-activating proteins (GAPs), feedback, and cell curvature. (a) GDP/GTP exchange factor (GEF) phosphorylation by p21-activated kinase (PAK) constitutes a negative feedback loop that limits GEF activity and hence the amount of GTP-Cdc42 in a polarity cluster. (b) A second negative feedback loop operates through septins and GAPs. (c) Cdc42-directed GAPs set a timescale for GTP hydrolysis, and the diffusion of GTP-Cdc42 at the membrane converts that timescale to a length scale that determines the lateral spread of GTP-Cdc42. Stronger GAP activity translates to a shorter lifetime and hence less lateral spread of active Cdc42. (d) Curvature of the cell membrane affects the size of the Cdc42 cluster in germinating spores of Schizosaccharomyces pombe, with higher curvature translating to a smaller cluster.

Figure 8

Landmarks and bud site selection in Saccharomyces cerevisiae. (a) Axl2, Bud8, and Bud9 are transmembrane proteins that are inherited at the indicated locations in newborn daughter cells and influence the location of polarity sites (red) in the next cell cycle. Haploid cells prefer Axl2-marked sites, and diploid cells prefer Bud8/Bud9-marked sites. These landmarks act by recruiting Bud5, the GDP/GTP exchange factor (GEF) for Rsr1, to locally generate GTP-Rsr1. GTP-Rsr1 in turn recruits Cdc24, the Cdc42-directed GEF. (b) The Cdc42-directed GTPase-activating protein (GAP) Rga1 blocks rebudding at the cytokinesis site, and the landmarks Rax1 and Rax2 anchor a memory complex at cytokinesis remnants that blocks polarization at all previous bud sites.