The tail of a yeast class V myosin, Myo2p, functions as a localization domain

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN ). Overexpression of the tail domain of Myo2p results in a dominant-negativ e phenotype that is phenotypically similar to a temperature-sensitive allel e of myo2, myo2-66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo 2p from sites of polarized cell growth. Subcellular fractionation of polari zed, mechanically lysed yeast cells reveals that Myo2p is present predomina ntly in a 100,000 x g pellet. The Myo2p in this pellet is not solubilized b y Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpre ssion does not disrupt this fractionation pattern, nor do mutations in sec4 , sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assemb ly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.