Smy1p: An Orphan Kinesin Finds a Home

Long distance cargo transport in budding yeast is carried out not by kinesin, but along actin cables by two non-processive class V myosins, Myo2p and Myo4p. Overexpression of Smy1p, a kinesin-related protein, rescues the temperature sensitive myo2-66 mutant yeast strain, which is defective in Myo2p transport.1 The mechanism by which a kinesin family protein rescues actin-based transport is unknown, but does not require microtubules.2 To address this question, we expressed Smy1p and Myo2p in insect cells and characterized them in vitro. Smy1p does not move microtubules in an ensemble motility assay, and is not an active motor. Using total internal reflection fluorescence microscopy (TIRFM), we find that Smy1p does not bind strongly to microtubules, but diffuses along them in the presence or absence of ATP. Surprisingly, Smy1p also binds to and diffuses along actin-fascin bundles. This binding is ionic strength-dependent, indicating the interaction is electrostatic in nature. When a single Myo2p is attached to a quantum dot cargo, the complex does not move processively on actin bundles. However, when several Smy1p molecules are attached to the quantum dot in addition to a single Myo2p, the complex supports continuous, unidirectional movement. 46% of moving quantum dots run to the end of the actin bundle, with run lengths greater than 10 microns observed. We hypothesize that Smy1p acts as an electrostatic tether, keeping the quantum dot bound to actin after Myo2p undergoes its powerstroke. We propose that overexpression of Smy1p rescues the myo2-66 mutant by enhancing the binding of cargo to actin. A similar mechanism likely contributes to transport in wild-type cells when both Smy1p and Myo2p are present on the same cargo.1. SH Lillie and SS Brown (1992), Nature 356, 358-61.2. SH Lillie and SS Brown (1998), JCB 140, 873-83.