The Saccharomyces cerevisiae bud-neck proteins Kcc4 and Gin4 have distinct but partially-overlapping cellular functions.

In the budding yeast S. cerevisiae, Swe1 delays the onset of mitosis by phosphorylation and inactivation of the cyclin-dependent kinase Cdc28, thereby relaying the morphogenetic signal to the cell cycle. Hsl1/Nik1, Kcc4 and Gin4 are structurally homologous protein kinases that localize to the bud neck and negatively regulate Swe1 by phosphorylation. We report here that Kcc4 and Gin4 have partially overlapping but essentially distinct cellular functions. Deletion of KCC4 had a similar effect to GIN4 deletion, causing moderate defects in bud formation at stationary phase; overexpression of Kcc4 inhibited cell growth. KCC4 showed functional interaction with GIN4 in cdc28 mutants, and both Kcc4 and Gin4 proteins physically interacted with Swe1 in vitro. However, unlike gin4delta cells, kcc4Delta cells were not elongated but multi-budded at stationary phase, and showed resistance to 0.04% SDS and 0.003% calcofluor white. In light of the observation that Kcc4 and Gin4 specifically associate with distinct septin proteins, we propose that the observed functional distinction between Kcc4 and Gin4 is due to differences in septin association partners.

[1]  M. Grunstein,et al.  A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae. , 1996, Genes & development.

[2]  R. Rothstein 18 – One-Step Gene Disruption in Yeast , 1989 .

[3]  D. Lew,et al.  Control of Swe1p degradation by the morphogenesis checkpoint , 1998, The EMBO journal.

[4]  Daniel J. Lew,et al.  A Morphogenesis Checkpoint Monitors the Actin Cytoskeleton in Yeast , 1998, The Journal of cell biology.

[5]  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.

[6]  L. Hartwell,et al.  Checkpoints: controls that ensure the order of cell cycle events. , 1989, Science.

[7]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[8]  M. Snyder,et al.  Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. , 1999, Genes & development.

[9]  S. Elledge,et al.  Gene identification using the yeast two-hybrid system. , 1997, Methods in enzymology.

[10]  J. Thorner,et al.  Hsl7 Localizes to a Septin Ring and Serves as an Adapter in a Regulatory Pathway That Relieves Tyrosine Phosphorylation of Cdc28 Protein Kinase inSaccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[11]  R. Altman,et al.  Control of Mitotic Events by Nap1 and the Gin4 Kinase , 1997, The Journal of cell biology.

[12]  J. Yates,et al.  Cell cycle-dependent assembly of a Gin4-septin complex. , 2002, Molecular biology of the cell.

[13]  David Lydall,et al.  The identification of a second cell cycle control on the HO promoter in yeast: Cell cycle regulation of SWI5 nuclear entry , 1990, Cell.

[14]  T. Hunter,et al.  The protein kinases of budding yeast: six score and more. , 1997, Trends in biochemical sciences.

[15]  L. Johnston,et al.  A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5 , 1993, Molecular and cellular biology.

[16]  J. Thorner,et al.  Dynamic localization of the Swe1 regulator Hsl7 during the Saccharomyces cerevisiae cell cycle. , 2001, Molecular biology of the cell.

[17]  R. Rothstein One-step gene disruption in yeast. , 1983, Methods in enzymology.

[18]  Indrani Bose,et al.  The Rho‐GAP Bem2p plays a GAP‐independent role in the morphogenesis checkpoint , 2002, The EMBO journal.

[19]  L. Johnston,et al.  DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Chandra L. Theesfeld,et al.  Determinants of Swe1p degradation in Saccharomyces cerevisiae. , 2002, Molecular biology of the cell.

[21]  H. Nojima,et al.  Nik1: a Nim1‐like protein kinase of S. cerevisiae interacts with the Cdc28 complex and regulates cell cycle progression , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[22]  D. Lew Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae. , 2000, Current opinion in genetics & development.

[23]  Y. Watanabe,et al.  A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C , 1993, Molecular and cellular biology.

[24]  H. Nojima,et al.  Gin4 of S. cerevisiae is a bud neck protein that interacts with the Cdc28 complex , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[25]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[26]  Chandra L. Theesfeld,et al.  Septin-Dependent Assembly of a Cell Cycle-Regulatory Module in Saccharomyces cerevisiae , 2000, Molecular and Cellular Biology.

[27]  M. Longtine,et al.  Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function , 1998, The Journal of cell biology.

[28]  M. Tyers,et al.  Regulation of cell cycle progression by Swe1p and Hog1p following hypertonic stress. , 2001, Molecular biology of the cell.

[29]  D. Lew,et al.  Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint , 1999, Molecular and Cellular Biology.

[30]  H Nojima,et al.  Kcc4 associates with septin proteins of Saccharomyces cerevisiae , 2001, FEBS letters.

[31]  Christopher W Carroll,et al.  The Septins Are Required for the Mitosis-specific Activation of the Gin4 Kinase , 1998, Journal of Cell Biology.

[32]  S. Reed,et al.  Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1. , 1998, Genes & development.

[33]  Y. Barral,et al.  Septins: a ring to part mother and daughter , 2002, Current Genetics.

[34]  Y. Ohya,et al.  A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint , 2001, Nature Cell Biology.

[35]  M. Snyder,et al.  Microtubule capture by the cleavage apparatus is required for proper spindle positioning in yeast. , 2002, Genes & development.