Abstract
We have investigated the minimal requirements of the tail region for myosin I function in vivo using the filamentous fungus Aspergillus nidulans. The CL3 strain (McGoldrick, C. A., Gruver, C., and May, G. S. (1995) J. Cell Biol. 128, 577-587) was transformed with a variety of myoA constructs containing mutations in the IQ, TH-1-like, SH3, and proline-rich domains by frameshift or in-frame deletions of the tail domains. The resulting strains contained wild type myoA driven by the alcA promoter and a mutant myoA driven by its endogenous promoter. This strategy allowed for selective expression of the wild type and/or mutant form of MYOA by the choice of growth medium. Proper septation and hyphal branching were found to be dependent on the interaction of the IQ motifs with calmodulin, as well as, the presence of its proline-rich domain. Additionally, a single proline-rich motif was sufficient for nearly wild type MYOA function. Most surprisingly, the SH3 domain was not essential for MYOA function. These studies expand our previous knowledge of the function of MYOA to include roles in hyphal morphogenesis, septal wall formation, and cell polarity, laying the groundwork for more detailed investigations on the function of the various tail domains in MYOA.
Highlights
Acanthamoeba castellani [8], and Aspergillus nidulans [9]
We previously showed that MYOA plays a role in secretion and polarized growth by the creation of a conditionally null myoA strain, CL3, in which the inducible alcohol dehydrogenase promoter drives the expression of myoA [9]
IQ-1/T3 myoA4/IQ-2 tail homology (TH)-1-like-1/T3 myoA4/TH-1-like-2 SH3–1/T3 myoA4/SH3–2 myoA4/T3 myoA4/T3 myoA4/T3 bp Detection of the myoA Frameshift and Deletion Mutations in the Genome—It is the tail region of class I myosins that is thought to provide them with their specific cellular location and function
Summary
Acanthamoeba castellani [8], and Aspergillus nidulans [9]. class I myosins have been studied in a variety of systems, the elucidation of their function has been complicated by the presence of multiple class I myosins with apparently overlapping function, such that loss of any single myosin I and in some cases deletion of multiple myosins I is not lethal. The discovery of myoA as an essential class I myosin in A. nidulans [9] allows us to address many aspects of MYOA function, including regulation by phosphorylation, protein interactions, and the functions of the specific domains present in the carboxyl terminus (COOH terminus). This paper addresses the in vivo significance of the four domains present in the COOH-terminal region of MYOA. We have chosen to mutate the tail domains of myoA because it is this portion of the polypeptide that leads to specific cellular localization and function. We have transformed six different mutant tail constructs corresponding to frameshift and deletion mutations and tested them for their ability to complement the conditional null phenotype of CL3. We have characterized the phenotypes of the viable myoA frameshift and deletion mutants by examining the consequences of these changes on cell growth, cell morphology, protein secretion, and endocytosis
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