Reconstituting a Native Actin Track for Myosin V Transport

Abstract

The budding yeast S. cerevisiae is an excellent model system for the study of cargo transport by myosin, given the relative importance of actin cables versus microtubules in this cell. Despite this, no in vitro studies have tried to mimic the actin-tropomyosin cables along which the class V myosin Myo2p transports secretory vesicles, vacuoles, mitochondria, and other organelles to the growing bud. We find that Myo2p is non-processive in vitro,in agreement with previous results 1-2. These experiments were, however, performed using chicken skeletal actin, which is only 87% identical to yeast actin. Accordingly, we are investigating if Myo2p behavior changes when the in vitro conditions more closely match those found in the yeast cell. Actin cables will be reconstituted in vitro from yeast actin, yeast tropomyosin, and the actin bundling protein fimbrin. The effects of yeast versus skeletal actin, of bundled actin versus single filaments, and of the presence of each of the two different tropomysin isoforms will be tested. Myo2p motility, processivity, and actin binding affinity will be assessed with these different tracks. The effect of varying ionic conditions, nucleotide concentration, and viscosity will also be tested, to determine if Myo2p behavior changes as conditions more closely match those of the intracellular milieu. 1. Reck-Peterson et al., JCB 153 (2001). 2. Dunn et al., JCB 178 (2007).