The RHO1‐GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase‐activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7, but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1‐controlled PKC1‐MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1‐mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1 GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.

[1]  M. Crouzet,et al.  Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae , 2001, FEBS letters.

[2]  Yoshikazu Ohya,et al.  Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3‐β‐glucan synthesis , 2001 .

[3]  Wei Guo,et al.  Spatial regulation of the exocyst complex by Rho1 GTPase , 2001, Nature Cell Biology.

[4]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[5]  D. E. Levin,et al.  Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1 , 2001, Molecular and Cellular Biology.

[6]  Y. Ohya,et al.  Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis. , 2001, Yeast.

[7]  International Human Genome Sequencing Consortium.  Initial sequencing and analysis of the human genome , 2001, Nature.

[8]  F. Klis,et al.  Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance. , 2000, Microbiology.

[9]  M. Molina,et al.  Regulatory Mechanisms for Modulation of Signaling through the Cell Integrity Slt2-mediated Pathway in Saccharomyces cerevisiae * , 2000, The Journal of Biological Chemistry.

[10]  Michael N. Hall,et al.  Cell Wall Stress Depolarizes Cell Growth via Hyperactivation of Rho1 , 1999, The Journal of cell biology.

[11]  Troy Ketela,et al.  Saccharomyces cerevisiae Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of thePKC1-MPK1 Cell Integrity Pathway , 1999, Journal of bacteriology.

[12]  Y. Ohya,et al.  The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton , 1998, Current Biology.

[13]  M. Bickle,et al.  Cell wall integrity modulates RHO1 activity via the exchange factor ROM2 , 1998, The EMBO journal.

[14]  L. Johnston,et al.  Analysis of RhoA-binding Proteins Reveals an Interaction Domain Conserved in Heterotrimeric G Protein β Subunits and the Yeast Response Regulator Protein Skn7* , 1998, The Journal of Biological Chemistry.

[15]  Peter Novick,et al.  Sec3p Is a Spatial Landmark for Polarized Secretion in Budding Yeast , 1998, Cell.

[16]  A. Hall,et al.  Rho GTPases and the actin cytoskeleton. , 1998, Science.

[17]  V. J. Cid,et al.  A mutation in the Rho1-GAP-encoding gene BEM2 of Saccharomyces cerevisiae affects morphogenesis and cell wall functionality. , 1998, Microbiology.

[18]  E. Cabib,et al.  Role of small G proteins in yeast cell polarization and wall biosynthesis. , 1998, Annual review of biochemistry.

[19]  M. Hall,et al.  Signaling to the actin cytoskeleton. , 1998, Annual review of cell and developmental biology.

[20]  L. Van Aelst,et al.  Rho GTPases and signaling networks. , 1997, Genes & development.

[21]  Marc Bickle,et al.  The Yeast Phosphatidylinositol Kinase Homolog TOR2 Activates RHO1 and RHO2 via the Exchange Factor ROM2 , 1997, Cell.

[22]  T Watanabe,et al.  Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae. , 1996, The EMBO journal.

[23]  L. Johnston,et al.  Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. , 1996, The EMBO journal.

[24]  Y. Anraku,et al.  Activation of Yeast Protein Kinase C by Rho1 GTPase (*) , 1996, The Journal of Biological Chemistry.

[25]  T Watanabe,et al.  Identification of Yeast Rho1p GTPase as a Regulatory Subunit of 1,3-β-Glucan Synthase , 1996, Science.

[26]  Guang-Chao Chen,et al.  Rho1p, a Yeast Protein at the Interface Between Cell Polarization and Morphogenesis , 1996, Science.

[27]  K. Tanaka,et al.  A downstream target of RHO1 small GTP‐binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. , 1995, The EMBO journal.

[28]  A. Bretscher,et al.  The rho-GAP encoded by BEM2 regulates cytoskeletal structure in budding yeast. , 1995, Molecular biology of the cell.

[29]  R. Cerione,et al.  Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast , 1994, The Journal of cell biology.

[30]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[31]  T. Dunn,et al.  Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae. , 1990, Molecular and cellular biology.

[32]  R. Axel,et al.  Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[34]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[35]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .