Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.

Ohya, Yoshikazu; Qadota, Hiroshi; Anraku, Yasuhiro; Pringle, John R.; Botstein, David

doi:10.1091/mbc.4.10.1017

Cited by 70 publications

(60 citation statements)

References 50 publications

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“…This result confirmed that Chs4p is prenylated in vivo. Since prenylation is an irreversible process, we assume that if Chs4p is preferentially farnesylated, inactivation of endogenous FTase will increase the amount of unmodified Chs4p available as a substrate for reaction in vitro, even if crossspecificity between FTase and GGTase I are observed (29,31,41). To prove the involvement of FTase in modification of Chs4p, we immobilized TAP-Chs4p expressed in wt yeast cells or cells lacking the catalytic subunit of FTase (Ram1p) on IgG-Sepharose beads and used the resin-Chs4p as a substrate for FTase assays in vitro.…”

Section: Resultsmentioning

confidence: 99%

“…3 show that chs4-C693S yeast cells harboring the nonprenylated version of Chs4p are more resistant to CFW than are wt cells and less resistant than are chs4⌬ yeast cells. To confirm the role of prenylation of Chs4p, we also constructed a chs4-I695L,M696L yeast strain expressing Chs4p with the C-terminal CVLL motif (the CVLL motif present in yeast Rho1p was proven experimentally to be a substrate for geranylgeranyl transferase type I [31]). Geranylgeranylation of Chs4p only partially restores the sensitivity to CFW.…”

Section: Vol 6 2007 Chitin Synthesis In S Cerevisiae 331mentioning

confidence: 99%

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Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2007

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Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ϳ60% decrease in CSIII activity, which is correlated with a ϳ30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

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Section: Resultsmentioning

confidence: 99%

Section: Vol 6 2007 Chitin Synthesis In S Cerevisiae 331mentioning

confidence: 99%

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2007

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“…Correct cellular targeting is essential for the function of Rho GTPases (Ohya et al, 1993). One aspect of Rho GTPase function is to localize effectors and other proteins required for polarization to the imminent growth site (Johnson, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…These include the proposed GTP-binding and hydrolysis domains (GDGAVGKT and DTAGQE), a GDP to GTP exchange domain (TKLD), a domain for effector interaction (TVFDNY), and a C terminal CAAX motif (where A indicates an aliphatic amino acid and X any amino acid), which is predicted to allow association with the plasma membrane after prenylation (Ohya et al, 1993;Ziman et al, 1991;Chen et al, 1993).…”

Section: Magnoportha Grisea (Af267176) and Suillus Bovinus (Af235004)mentioning

confidence: 99%

Control of morphogenesis and actin localization by thePenicillium marneffei RAChomolog

Boyce

Hynes

Andrianopoulos

2003

Journal of Cell Science

124

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Rac proteins control polarized growth in many organisms but the specific function of these proteins remains undefined. In this study, we describe the cloning and functional characterization of a RAC homolog, cflB, from the dimorphic fungus Penicillium marneffei. P. marneffei produces asexual spores on complex structures (conidiophores)and switches between hyphal and yeast growth. CflB colocalizes with actin at the tips of vegetative hyphal cells and at sites of cell division. Deletion of cflB results in cell division (septation) and growth defects in both vegetative hyphal and conidiophore cell types such that cells become depolarized, exhibit inappropriate septation and the actin cytoskeleton is severely disrupted. This data suggests that Rac proteins play a crucial role in actin dependent polarized growth and division. The CDC42 ortholog in P. marneffei, cflA, controls vegetative hyphal and yeast growth polarization but does not affect asexual development. By contrast, CflB affects cellular polarization during asexual development and hyphal growth but not during yeast growth. This shows that these two GTPases have both overlapping and distinct roles during growth and development. RAC orthologs are not found in less morphologically complex eukaryotes such as Saccharomyces cerevisiae, suggesting that RAC genes might have evolved with increasing cellular complexity.

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“…Although GGTase I normally is an essential gene for growth, it can be made non-essential, whenthe dosage of the two GTPases, Rholp (30) and Cdc42p (22), are artificially elevated (41). Since the yeast GGTase I prenylates these two GTPases, Cdc42p and Rholp are implicated genetically as the two essential substrates of GGTase I (41).…”

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confidence: 99%

Signaling toward Yeast 1,3-.BETA.-glucan Synthesis.

Inoue

Qadota

Arisawa

et al. 1996

Cell Struct. Funct.

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Key words: yeast/cell wall/l ,3-/3-glucan synthase/rho GTPase/geranylgeranyltransferase I/plasma membraneinvagination ABSTRACT. 1,3-^glucan synthase catalyzes the synthesis of a 1,3-^-linked glucan polymer which produces the main rigidity of the yeast cell wall. Recent success in purification of this enzyme by product entrapment (21) has provided new insights into the dynamic aspects of the cell wall. This relatively simple procedure made it possible to identify the genes encoding the catalytic subunits of glucan synthase. In addition, the involvement of a rho type GTPase in the regulation of glucan synthase was demonstrated with the purified enzyme. Based on intracellular localization of the glucan synthase subunits, we have proposed a model in which assembly of the subunits is important for the activation of glucan synthase at sites of polarized growth. In this article, we will focus on biochemistry of 1,3-^-glucan synthase and signaling through rho type GTPase.The yeast cell wall is not a static structure. During cell growth, cell wall precursors are polymerized at the cell surface, then modified and connected to each other.Other materials are madein the endomembranesystem, secreted to the cell surface and subsequently assembled into the cell wall. After the primary cell wall is formed, further modification and deposition of wall components frequently occur. Comparedwith the mature cell wall, the newly-synthesized cell wall is unable to provide full protection from mechanical or chemical stress. However, the synthesis and insertion of new cell wall is not normally deleterious to cell growth. That the new cell wall is not a liability to cell growth suggests that synthesis and assembly of the cell wall are well coordinated and occurs in such a wayas to reduce the effects of mechanical and chemical stress. This coordinate synthesis and assembly is most necessary at the onset of the cell surface growth when, in the presence of high turgor pressure, the existing cell wall must be loosened enzyma- mAb, monoclonal antibody; SDS-PAGE, polyacrylamide gel electrophoresis with SDS; UDPG, UDP-glucose.

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Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.

Cited by 70 publications

References 50 publications

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

Control of morphogenesis and actin localization by thePenicillium marneffei RAChomolog

Signaling toward Yeast 1,3-.BETA.-glucan Synthesis.

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