Cortical Num1p Interacts with the Dynein Intermediate Chain Pac11p and Cytoplasmic Microtubules in Budding Yeast

Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the α-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p–Kar9p complex at the bud tip of early anaphase cells.

[1]  M. Rose,et al.  Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. , 2000, Molecular biology of the cell.

[2]  M. Hoyt,et al.  Mitotic motors in Saccharomyces cerevisiae. , 2000, Biochimica et biophysica acta.

[3]  M. Rose,et al.  The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization , 1999, The Journal of cell biology.

[4]  D. Botstein,et al.  BIM1 encodes a microtubule-binding protein in yeast. , 1997, Molecular biology of the cell.

[5]  J. Cooper,et al.  Microtubule Interactions with the Cell Cortex Causing Nuclear Movements in Saccharomyces cerevisiae , 2000, The Journal of cell biology.

[6]  L. Frisén,et al.  The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast. , 1998, Molecular biology of the cell.

[7]  S. Osmani,et al.  NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. , 1995, Molecular biology of the cell.

[8]  I R Gibbons,et al.  Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation , 1995, The Journal of cell biology.

[9]  P. Slonimski,et al.  Chemotyping of yeast mutants using robotics , 1999, Yeast.

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

[11]  Sabine Martin,et al.  Phospholipase C Binds to the Receptor-like GPR1Protein and Controls Pseudohyphal Differentiation inSaccharomyces cerevisiae * , 1999, The Journal of Biological Chemistry.

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

[13]  J. Cooper,et al.  Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin. , 2000, Molecular biology of the cell.

[14]  D. Ledbetter,et al.  Isolation of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like repeats. , 1993, Nature.

[15]  J. Schlessinger,et al.  PH Domains: Diverse Sequences with a Common Fold Recruit Signaling Molecules to the Cell Surface , 1996, Cell.

[16]  M. Aigle,et al.  The NUM1 yeast gene: Length polymorphism and physiological aspects of mutant phenotype , 1993, Yeast.

[17]  M. Farkašovský,et al.  Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions , 1995, The Journal of cell biology.

[18]  R. Vallee,et al.  A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function , 2000, Nature Cell Biology.

[19]  Charles Boone,et al.  Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p , 1999, The Journal of cell biology.

[20]  M. Kikyo,et al.  Bni1p Regulates Microtubule-Dependent Nuclear Migration through the Actin Cytoskeleton in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[21]  S. Karki,et al.  Cytoplasmic dynein and dynactin in cell division and intracellular transport. , 1999, Current opinion in cell biology.

[22]  N. Cole,et al.  Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways. , 1997, Molecular biology of the cell.

[23]  M. Rose,et al.  Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast , 1998, The Journal of cell biology.

[24]  Thomas Fiedler,et al.  A new efficient gene disruption cassette for repeated use in budding yeast , 1996, Nucleic Acids Res..

[25]  D. Pellman,et al.  Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration , 1997, The Journal of cell biology.

[26]  B. Suter,et al.  Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning , 1999, Nature Cell Biology.

[27]  A. Chaudhuri,et al.  Molecular linkage underlying microtubule orientation toward cortical sites in yeast. , 2000, Science.

[28]  J. Cooper,et al.  A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration , 1994, Cell.

[29]  D. Pellman,et al.  Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. , 2000, Science.

[30]  K. Bloom,et al.  The Role of Actin in Spindle Orientation Changes during the Saccharomyces cerevisiae Cell Cycle , 1999, The Journal of cell biology.

[31]  M. Hoyt,et al.  Mitotic Spindle Positioning in Saccharomyces cerevisiae Is Accomplished by Antagonistically Acting Microtubule Motor Proteins , 1997, The Journal of cell biology.

[32]  E. O'Toole,et al.  Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules , 1999, The Journal of cell biology.

[33]  R. Heald,et al.  Microtubule-based motor function in mitosis. , 1999, Current opinion in structural biology.

[34]  S. Shaw,et al.  Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud , 1997, The Journal of cell biology.

[35]  S. Shaw,et al.  Nuclear and spindle dynamics in budding yeast. , 1998, Molecular biology of the cell.

[36]  S Falkow,et al.  Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. , 1997, Microbiology.

[37]  P. Silver,et al.  The yeast dynactin complex is involved in partitioning the mitotic spindle between mother and daughter cells during anaphase B. , 1998, Molecular biology of the cell.

[38]  G. Boguslawski PBS2, a yeast gene encoding a putative protein kinase, interacts with the RAS2 pathway and affects osmotic sensitivity of Saccharomyces cerevisiae. , 1992, Journal of general microbiology.

[39]  L. Tsai,et al.  Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1 , 2000, Nature Cell Biology.

[40]  Tim Stearns,et al.  Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex , 1997, The Journal of cell biology.