Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.

Ziman, Michael; Preuss, Daphne; Mulholland, Jon; O'Brien, Jeanne M.; Botstein, David; Johnson, Douglas I.

doi:10.1091/mbc.4.12.1307

Cited by 246 publications

(243 citation statements)

References 38 publications

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“…Failure of temperature-sensitive cdc24 and cdc42 mutations to form buds implicated Cdc24p and Cdc42p in bud organization (16), an idea supported in the case of Cdc42p by its localization in the growing tip of the bud (20). The amino acid sequences of Bem3p and Cdc24p showed them to be homologous to GTPase-activating proteins and guanine nucleotideexchange factors, respectively, a result confirmed by direct biochemical analysis (21).…”

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confidence: 83%

Section: Introductionmentioning

confidence: 99%

“…RH02 is not essential for growth under any conditions tested (13). Of these rho-homologous genes, Cdc42p has been best studied to date both genetically and biochemically (16)(17)(18)(19)(20).The functions of some of the rho subfamily proteins have been investigated by using conditional-lethal mutations. Failure of temperature-sensitive cdc24 and cdc42 mutations to form buds implicated Cdc24p and Cdc42p in bud organization (16), an idea supported in the case of Cdc42p by its localization in the growing tip of the bud (20).…”

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confidence: 99%

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Conditional lethality of a yeast strain expressing human RHOA in place of RHO1.

Qadota

Anraku²,

Botstein³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The yeast RHO1 GTPase, which has 72% amino acid sequence identity with its human counterpart, RHOA, is essential for growth, although the reason has not been investigated. We report here that yeast strains that rely solely on expression of human RHOA in place ofRHOI are able to grow at 23C but grow neither at 37C nor in the presence of 300 mM CaCl2 even at 23C. Measurements of steady-state protein levels indicate that inability to grow at the restrictive temperature is not due to instability of the protein. Homolog scanning with the two GTPases identified a small, 27-residue region of RHO) which, when substituted into RHOA, confers full function in yeast. This region corresponds to the a3-helix loop 7 region of RAS; the same region was reported to determine specificity of function between GTPases of the RAB family, Sec4p and Yptlp. By examining the phenotype of RHOA substitution strains at nonpermissive temperature, we found evidence suggesting that the normal function ofRHOI is to maintain osmotic Integrity.Rho GTPases, which comprise one subfamily of ras-related GTP-binding proteins, are thought to act as molecular switches by cycling between two states (the active GTPbound form and the inactive GDP-bound form) (1,2). Transitions between these two states are controlled by regulator molecules, including GTPase-activating proteins, guanine nucleotide-exchange factors, and guanine nucleotidedissociation inhibitors. Many such regulator molecules have been identified for mammalian rho GTPases (3). Evidence is accumulating that the rho family GTPases function in cell morphology (4), cell motility (5), cell adhesion (6, 7), cytokinesis (8), and smooth muscle contraction (9) through regulation of actin assembly (10-12).In the yeast Saccharomyces cerevisiae, five members of the rho subfamily (RHO), RHO2, RHO3, RHO4, and CDC42) have been identified (13-17). RHO) and CDC42 are essential genes (13,17). RHO3 and RHO4 are related functionally to each other and loss ofboth results in growth failure above 300C (14,15). RH02 is not essential for growth under any conditions tested (13). Of these rho-homologous genes, Cdc42p has been best studied to date both genetically and biochemically (16)(17)(18)(19)(20).The functions of some of the rho subfamily proteins have been investigated by using conditional-lethal mutations. Failure of temperature-sensitive cdc24 and cdc42 mutations to form buds implicated Cdc24p and Cdc42p in bud organization (16), an idea supported in the case of Cdc42p by its localization in the growing tip of the bud (20). The amino acid sequences of Bem3p and Cdc24p showed them to be homologous to GTPase-activating proteins and guanine nucleotideexchange factors, respectively, a result confirmed by direct biochemical analysis (21). In contrast, little is known about the function of RHO) since no conditional-lethal mutants have yet been reported. A possible function of RHO) in the secretory process was suggested by the observation that Rholp is found associated with the Golgi apparatus and post-Golgi ves...

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confidence: 83%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Conditional lethality of a yeast strain expressing human RHOA in place of RHO1.

Qadota

Anraku²,

Botstein³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Cdc42p is localized to the presumptive bud site early in the cell cycle (Ziman et al, 1993;Richman et al, 2002) and controls the initial formation of the septin ring at that stage. Cdc42p is also localized to the mother-bud neck late in the cell cycle (Richman et al, 2002) and thus might play an additional role in maintaining septin organization (or promoting reorganization) at that stage.…”

Section: Localization Of Cdc42p Gaps In the Cell Cyclementioning

confidence: 99%

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Caviston

Longtine

Pringle

et al. 2003

MBoC

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The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42 V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42 V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth. INTRODUCTIONThe septin family of proteins was first recognized in yeast and now appears to be ubiquitous in fungi and animals, although not in plants (Longtine et al., 1996;Field and Kellogg, 1999;Trimble, 1999;Nguyen et al., 2000;Momany et al., 2001;Macara et al., 2002). Typical septins have a variable N-terminal region, a conserved core that includes the elements of a GTP-binding site, and a variable C-terminal region that (in all but a few cases) includes a predicted coiled-coil domain. In each species examined to date, there are two or more septins that form heterooligomeric complexes to perform their specific functions. Purified septins from yeast, Drosophila, Xenopus, and mammalian cells form filaments of 7-9 nm diameter and of variable length (Field et al., 1996;Frazier et al., 1998;Hsu et al...

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“…The rho-GTPases Cdc42p and Rho1p also localize to growth sites (McCaffrey et al, 1991;Ziman et al, 1993;Yamochi et al, 1994) and interact with Bni1p in two-hybrid assays (Kohno et al, 1996;Evangelista et al, 1997). Cdc42p is required for recruitment of Bni1p to the nascent bud site (Jaquenoud and Peter, 2000;Ozaki-Kuroda et al, 2001) and when overexpressed, Cdc42p promotes Bni1p-dependent cable assembly (Dong et al, 2003).…”

Section: Rho-gtpases Depend On Polarized Secretion For Bud Tipassociamentioning

confidence: 99%

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

Pruyne

Gao

et al. 2004

MBoC

151

208

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Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell. INTRODUCTIONProper cell polarization requires a balance between dynamism and stability. Persistent systems, such as the apical and basolateral domains of epithelial cells, show a higher stability than flexible systems, such as cells undergoing chemotaxis. However, both types can use similar cytoskeletal components, so an important task in understanding the control of polarity is to identify features that influence persistence, and how those features integrate with the core cytoskeletal machinery providing the physical expression of polarity.The process of bud growth of the yeast Saccharomyces cerevisiae provides a model for examining the control of polarity (for reviews, see Bretscher, 2003;Chang and Peter, 2003). Secretory organelles, such as the Golgi and endoplasmic reticulum, are distributed throughout the bud and mother cell, whereas post-Golgi secretory vesicles concentrate at discrete growth sites at the cell cortex. These vesicles are guided to these sites along what are essentially two axes of polarity: a stable axis directing traffic from the mother into the bud, and a dynamic axis that fine-tunes delivery from the bud neck to specific regions in the bud, sending vesicles first to the small bud tip, then to the entire bud surface, and finally back toward the neck during mother/daughter separation. On delivery to these locations, post-Golgi vesicles fuse with the cell surface to promote localized cell expansion.Two class-V myosins, with heavy chains encoded by MYO2 (Johnston et al., 1991) and MYO4 (Haarer et al., 1994), propel cellular components, including vesicles, organelles, mRNAs, and microtubules, from the mother into the bud during growth and organelle segregation. The myosins move along cables of actin filaments that radiate throughout the cell in arrays oriented toward growth sites (Adams and Pringle, 1984;Kilmartin and Adams, 1984).Assembly of these cables depends on two formin homologues, Bni1p and Bnr1p (Evangelista et al., 2002;Sagot et al., 2002a), members of a family of cytoskeletal regulatory proteins defined by conserved fo...

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Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.

Cited by 246 publications

References 38 publications

Conditional lethality of a yeast strain expressing human RHOA in place of RHO1.

Conditional lethality of a yeast strain expressing human RHOA in place of RHO1.

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

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