The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Δ myo5Δ cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A–induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37°C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Δ myo5Δ cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Δ myo5Δ cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.
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15 June 1998
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June 15 1998
The Src Homology Domain 3 (SH3) of a Yeast Type I Myosin, Myo5p, Binds to Verprolin and Is Required for Targeting to Sites of Actin Polarization
Blake L. Anderson,
Blake L. Anderson
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
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Istvan Boldogh,
Istvan Boldogh
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
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Marie Evangelista,
Marie Evangelista
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
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Charles Boone,
Charles Boone
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
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Lloyd A. Greene,
Lloyd A. Greene
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
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Liza A. Pon
Liza A. Pon
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
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Blake L. Anderson
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
Istvan Boldogh
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
Marie Evangelista
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
Charles Boone
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
Lloyd A. Greene
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
Liza A. Pon
*Department of Pathology and ‡Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York 10032; and §Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
Address all correspondence to Liza A. Pon, Department of Anatomy and Cell Biology, Columbia University P&S 12-425, 630 West 168th Street, New York, NY 10032. Tel.: (212) 305-1947. Fax: (212) 305-3970. E-mail: [email protected]
Received:
October 28 1997
Revision Received:
April 23 1998
Online ISSN: 1540-8140
Print ISSN: 0021-9525
1998
J Cell Biol (1998) 141 (6): 1357–1370.
Article history
Received:
October 28 1997
Revision Received:
April 23 1998
Citation
Blake L. Anderson, Istvan Boldogh, Marie Evangelista, Charles Boone, Lloyd A. Greene, Liza A. Pon; The Src Homology Domain 3 (SH3) of a Yeast Type I Myosin, Myo5p, Binds to Verprolin and Is Required for Targeting to Sites of Actin Polarization . J Cell Biol 15 June 1998; 141 (6): 1357–1370. doi: https://doi.org/10.1083/jcb.141.6.1357
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