Studies on SMY1P and MYO2P, members of two motor protein families in Saccharomyces cerevisiae.

Abstract

Smy1p, a kinesin-related protein in Saccharomyces cerevisiae, and Myo2p, a class V myosin, have previously been linked by genetic and colocalization studies (Lillie, S. H., and S. S. Brown. 1992. Nature (Lond.). 356:358-361; Lillie, S. H., and S. S. Brown. 1994. J. Cell Bio. 125:825-842). The specific function of these proteins is unknown. In this dissertation two-hybrid analysis was used to determine that they interact physically. This is the first observation of a member of the kinesin superfamily of microtubule based motors physically interacting with an actin based myosin. Furthermore, the site of interaction has been mapped. Deletion of a region of the Smy1p tail eliminates its ability to localize and to overcome the myo2-66 mutant defects. An interaction between Myo2p molecules is also documented, indicating that they dimerize. Mutation of a potential phosphorylation site just downstream of the Myo2p coiled-coil region does not affect either dimerization or interaction with Smy1p. Localization studies have determined that like myo2-2p, Smy1p localization is lost in the myo2-2 mutant. Overexpression of Smy1p partially restores myo2-2p localization but requires the myosin binding region of its tail domain to do so. Other studies involve the use of a Smy1p affinity column to isolate Smy1p binding partners. The yeast killer virus was isolated from this column and was tested for transmission defects in a Smy1p deletion strain. In a third set of experiments, a series of motor protein chimeras were made and tested for their ability to function in vivo. Based on these results and previous studies it is concluded that Smy1p and Myo2p are functioning together possibly for the transport of secretory vesicles. Furthermore, these observations may serve as a prototype for the theory of sequential transport (the idea that microtubules are used for long distance transport and that cargo is passed on to the actin based motor for local delivery) in higher eukaryotes.