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J. Cell Biol.,
Volume 141, Number 6, June 15, 1998 1357-1370
* Department of Pathology and 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 myo3 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
myo5 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 myo3
myo5 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, myo3 myo5 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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