A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast.

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

[1]  L. Alphey,et al.  Vectors for the expression of tagged proteins in Drosophila. , 2001, BioTechniques.

[2]  T. Pollard,et al.  The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes , 1999, Current Biology.

[3]  Rong Li,et al.  Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Gary G. Borisy,et al.  Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia , 1999, The Journal of cell biology.

[5]  Matthew D. Welch,et al.  The Wiskott–Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex , 1999, Current Biology.

[6]  Rong Li,et al.  Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein , 1999, Current Biology.

[7]  Amanda Y. Chan,et al.  Relationship between Arp2/3 Complex and the Barbed Ends of Actin Filaments at the Leading Edge of Carcinoma Cells after Epidermal Growth Factor Stimulation , 1999, The Journal of cell biology.

[8]  M. Kirschner,et al.  The Interaction between N-WASP and the Arp2/3 Complex Links Cdc42-Dependent Signals to Actin Assembly , 1999, Cell.

[9]  T. Pollard,et al.  Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  K L Gould,et al.  The Arp2/3 complex: a multifunctional actin organizer. , 1999, Current opinion in cell biology.

[11]  E. Egelman,et al.  A change in actin conformation associated with filament instability after Pi release. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  T. Schroer,et al.  Actin-related proteins. , 1999, Annual review of cell and developmental biology.

[13]  Laura M. Machesky,et al.  Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex , 1998, Current Biology.

[14]  A. Carr,et al.  Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. , 1998, Gene.

[15]  D. Drubin,et al.  The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo , 1998, The Journal of cell biology.

[16]  T. Mitchison,et al.  Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. , 1998, Science.

[17]  P. Philippsen,et al.  Heterologous modules for efficient and versatile PCR‐based gene targeting in Schizosaccharomyces pombe , 1998, Yeast.

[18]  K. Gould,et al.  Isolation and characterization of new fission yeast cytokinesis mutants. , 1998, Genetics.

[19]  T D Pollard,et al.  The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  T. Pollard,et al.  Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments. , 1998, Molecular biology of the cell.

[21]  K. Ayscough,et al.  In vivo functions of actin-binding proteins. , 1998, Current opinion in cell biology.

[22]  K. Gould,et al.  The control of septum formation in fission yeast. , 1997, Genes & development.

[23]  A. Burlingame,et al.  Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins. , 1997, The Biochemical journal.

[24]  Gary G. Borisy,et al.  Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation , 1997, The Journal of cell biology.

[25]  E. Egelman,et al.  Modulation of yeast F-actin structure by a mutation in the nucleotide-binding cleft. , 1997, Journal of molecular biology.

[26]  A. DePace,et al.  The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly , 1997, The Journal of cell biology.

[27]  M. Mann,et al.  The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches , 1997, Current Biology.

[28]  V. Moreau,et al.  The yeast actin-related protein Arp2p is required for the internalization step of endocytosis. , 1997, Molecular biology of the cell.

[29]  L. Machesky,et al.  Cell motility: Complex dynamics at the leading edge , 1997, Current Biology.

[30]  Walter F. Stafford,et al.  Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba , 1997, The Journal of cell biology.

[31]  Timothy J. Mitchison,et al.  Actin polymerization is induced by Arp 2/3 protein complex at the surface of Listeria monocytogenes , 1997, Nature.

[32]  T. Mitchison,et al.  Actin dynamics in vivo. , 1997, Current opinion in cell biology.

[33]  K. Gould,et al.  Cytokinesis in fission yeast Schizosaccharomyces pombe. , 1997, Methods in enzymology.

[34]  K. Gould,et al.  Fission yeast Sop2p: a novel and evolutionarily conserved protein that interacts with Arp3p and modulates profilin function. , 1996, The EMBO journal.

[35]  K. Gould,et al.  The Schizosaccharomyces pombe actin‐related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin. , 1996, The EMBO journal.

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

[37]  B. Dujon,et al.  The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton , 1996, The Journal of cell biology.

[38]  T. Schroer,et al.  Actin-related protein 1 and cytoplasmic dynein-based motility - what's the connection? , 1996, Trends in cell biology.

[39]  T. Pollard,et al.  Actin' like actin? , 1996, Trends in cell biology.

[40]  D. Botstein,et al.  Movement of yeast cortical actin cytoskeleton visualized in vivo. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A. Wach.  PCR‐synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae , 1996, Yeast.

[42]  J. Cooper,et al.  Movement of cortical actin patches in yeast , 1996, The Journal of cell biology.

[43]  T D Pollard,et al.  Sequences, structural models, and cellular localization of the actin- related proteins Arp2 and Arp3 from Acanthamoeba , 1995, The Journal of cell biology.

[44]  X. Chen,et al.  The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides , 1995, Journal of Biological Chemistry.

[45]  A. Willems,et al.  Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure , 1995, Yeast.

[46]  P. Morandini,et al.  An actin‐related protein from Dictyostelium discoideum is developmentallyregulated and associated with mitochondria , 1995, FEBS letters.

[47]  J. Demeter,et al.  A mutation in the RCC1-related protein pim1 results in nuclear envelope fragmentation in fission yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[48]  W. Kabsch,et al.  The actin fold , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[49]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[50]  T. Pollard,et al.  Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose , 1994, The Journal of cell biology.

[51]  J. Boeke,et al.  Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. , 1994, Genetics.

[52]  D. Drubin,et al.  The yeast actin cytoskeleton. , 1994, Current opinion in cell biology.

[53]  B. Neel,et al.  Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. , 1993, Analytical biochemistry.

[54]  K. Maundrell,et al.  TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. , 1993, Gene.

[55]  P. Janmey,et al.  Cofilin is an essential component of the yeast cortical cytoskeleton , 1993, The Journal of cell biology.

[56]  C. Schutt,et al.  The structure of crystalline profilin-beta-actin. , 1993, Nature.

[57]  D. Drummond,et al.  The binding of mutant actins to profilin, ATP and DNase I. , 1992, European journal of biochemistry.

[58]  D. Helfman,et al.  A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility , 1992, Nature.

[59]  D. Nathans,et al.  Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[60]  W. Kabsch,et al.  Structure and function of actin. , 1992, Annual review of biophysics and biomolecular structure.

[61]  Jonathan A. Cooper,et al.  Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein , 1992, The Journal of cell biology.

[62]  A. Carr,et al.  Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe. , 1992, Gene.

[63]  H. Prentice,et al.  High efficiency transformation of Schizosaccharomyces pombe by electroporation. , 1992, Nucleic acids research.

[64]  E. Schwob,et al.  New yeast actin-like gene required late in the cell cycle , 1992, Nature.

[65]  K. Gould,et al.  Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function. , 1991, The EMBO journal.

[66]  D. Drummond,et al.  Stability of mutant actins. , 1991, The Biochemical journal.

[67]  S. Moreno,et al.  Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. , 1991, Methods in enzymology.

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

[69]  W. Kabsch,et al.  Atomic structure of the actin: DNase I complex , 1990, Nature.

[70]  W. Kabsch,et al.  Atomic model of the actin filament , 1990, Nature.

[71]  L. Szilágyi,et al.  Photoaffinity labeling of the nucleotide binding site of actin. , 1986, Biochemistry.

[72]  J. Marks,et al.  Growth Polarity and Cytokinesis in Fission Yeast: The Role of the Cytoskeleton , 1986, Journal of Cell Science.

[73]  B. Bormann,et al.  The photoaffinity probe 8‐N3[α‐32P]ATP labels the ATP‐binding sites of rabbit neutrophil and skeletal muscle actin , 1983, FEBS letters.

[74]  J. Olmsted,et al.  Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. , 1981, The Journal of biological chemistry.