Identification and functional analysis of the essential and regulatory light chains of the only type II myosin Myo1p in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p–Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p–Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes.

[1]  Rong Li,et al.  Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis , 1998, The Journal of cell biology.

[2]  P. Thuriaux,et al.  Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe , 1976, Molecular and General Genetics MGG.

[3]  J. Spudich,et al.  A functional recombinant myosin II lacking a regulatory light chain-binding site. , 1993, Science.

[4]  Richard A. Cerione,et al.  Iqg1p links spatial and secretion landmarks to polarity and cytokinesis , 2002, The Journal of cell biology.

[5]  Dannel McCollum,et al.  The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe. , 2002, Molecular biology of the cell.

[6]  M. Longtine,et al.  Regulation of septin organization and function in yeast. , 2003, Trends in cell biology.

[7]  C. Brenner,et al.  Yeast myosin light chain, Mlc1p, interacts with both IQGAP and class II myosin to effect cytokinesis. , 2000, Journal of cell science.

[8]  V. Simanis,et al.  The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p. , 2000, Journal of cell science.

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

[10]  I. Herskowitz,et al.  Osmotic Balance Regulates Cell Fusion during Mating in Saccharomyces cerevisiae , 1997, The Journal of cell biology.

[11]  Christopher G. Burd,et al.  The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization , 2003, The Journal of cell biology.

[12]  E. Bi Cytokinesis in budding yeast: the relationship between actomyosin ring function and septum formation. , 2001, Cell structure and function.

[13]  J. Chant,et al.  Cyk3, a novel SH3-domain protein, affects cytokinesis in yeast , 2000, Current Biology.

[14]  L. McIntosh,et al.  Structure of Cdc4p, a Contractile Ring Protein Essential for Cytokinesis in Schizosaccharomyces pombe * , 2001, The Journal of Biological Chemistry.

[15]  E. Bi,et al.  ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[16]  K. Trybus Regulation of smooth muscle myosin. , 1991, Cell motility and the cytoskeleton.

[17]  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.

[18]  Rong Li,et al.  Dual Function of Cyk2, a cdc15/PSTPIP Family Protein, in Regulating Actomyosin Ring Dynamics and Septin Distribution , 1998, The Journal of cell biology.

[19]  Daniel P. Mulvihill,et al.  Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring. , 2001, Journal of cell science.

[20]  F. Matsumura,et al.  Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells , 1998, The Journal of cell biology.

[21]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[22]  J. Caviston,et al.  Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae. , 2000, Molecular biology of the cell.

[23]  Daniel J. Lew,et al.  Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis , 1998, The Journal of cell biology.

[24]  R. Chisholm,et al.  The dictyostelium essential light chain is required for myosin function , 1992, Cell.

[25]  S. Silverman,et al.  The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle , 1991, The Journal of cell biology.

[26]  T. Davis,et al.  Mlc 1 p Is a Light Chain for the Unconventional Myosin Myo 2 p in Saccharomyces cerevisiae , 1998 .

[27]  J. Pringle,et al.  The septin cortex at the yeast mother-bud neck. , 2001, Current opinion in microbiology.

[28]  T. E. Schroeder The contractile ring. II. Determining its brief existence, volumetric changes, and vital role in cleaving Arbacia eggs. , 1972 .

[29]  Nicola Tolliday,et al.  Direct evidence for a critical role of myosin II in budding yeast cytokinesis and the evolvability of new cytokinetic mechanisms in the absence of myosin II. , 2003, Molecular biology of the cell.

[30]  K. Gould,et al.  Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis , 1995, The Journal of cell biology.

[31]  L. Hartwell,et al.  Genetic control of the cell division cycle in yeast. 3. Seven genes controlling nuclear division. , 1971, Experimental cell research.

[32]  S. Fields,et al.  A protein interaction map for cell polarity development , 2001, The Journal of cell biology.

[33]  Gary D Bader,et al.  Global Mapping of the Yeast Genetic Interaction Network , 2004, Science.

[34]  Claudio De Virgilio,et al.  A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall , 1997, The Journal of cell biology.

[35]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[36]  E. Salmon,et al.  Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae , 1995, The Journal of cell biology.

[37]  M. Umikawa,et al.  Interaction of Bnr1p with a Novel Src Homology 3 Domain-containing Hof1p , 1998, The Journal of Biological Chemistry.

[38]  H. Hosoya,et al.  Spatial localization of mono-and diphosphorylated myosin II regulatory light chain at the leading edge of motile HeLa cells. , 2002, Cell structure and function.

[39]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[40]  K. Edwards,et al.  The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila. , 1991, Cell.

[41]  C. Burd,et al.  A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast. , 1997, Molecular biology of the cell.

[42]  J. Caviston,et al.  The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast. , 2003, Molecular biology of the cell.

[43]  T. E. Schroeder THE CONTRACTILE RING , 1970, Zeitschrift für Zellforschung und Mikroskopische Anatomie.

[44]  P. Bielli,et al.  Mlc1p promotes septum closure during cytokinesis via the IQ motifs of the vesicle motor Myo2p , 2002, The EMBO journal.

[45]  Rong Li,et al.  A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation , 2000, Current Biology.

[46]  E. Bi,et al.  Cytokinesis: an emerging unified theory for eukaryotes? , 1999, Current opinion in cell biology.

[47]  T. Davis,et al.  Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae , 1998, The Journal of cell biology.

[48]  E. Cabib,et al.  In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each other. , 2002, Journal of cell science.

[49]  J. Sellers Regulation of cytoplasmic and smooth muscle myosin. , 1991, Current opinion in cell biology.

[50]  J. Chant,et al.  An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast , 1997, Current Biology.

[51]  D. Burgess,et al.  Targeted new membrane addition in the cleavage furrow is a late, separate event in cytokinesis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[52]  Roberto Dominguez,et al.  Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs—functional implications , 2003, The EMBO journal.

[53]  M. Balasubramanian,et al.  Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function , 2000, Nature Cell Biology.

[54]  J. Caviston,et al.  The Role of Cdc 42 p GTPase-activating Proteins ( GAPs ) in Assembly of the Septin Ring in Yeast , 2003 .

[55]  R. Chisholm,et al.  Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development , 1994, The Journal of cell biology.

[56]  H. Wang,et al.  Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe. , 2001, Cell structure and function.