Essential Features of a Non-processive Class V Myosin from Budding Yeast for ASH1 mRNA Transport

Abstract

A feature of most class V myosins is their ability to move processively on actin. The budding yeast Saccharomyces cerevisiae has a non-processive class V myosin, Myo4p, which is a single-headed but high duty cycle motor. Its cellular role is to asymmetrically transport more than 20 different mRNAs, a widely used strategy to polarize a protein within the cell. The most studied mRNA is ASH1, which is moved by Myo4p to the bud tip to repress mating type switching in the daughter cell. Here we determine the features of Myo4p that are necessary for correct localization of ASH1 mRNA to the daughter cell. This process requires the adapter protein She3p, and the mRNA binding protein She2p, which binds ASH1 at specific localization elements called zip codes. Based on a series of chimeric constructs, we showed that the rod region of Myo4p, but not the globular tail, is essential for correct localization of ASH1 mRNA. The rod thus contains the primary binding site for She3p, consistent with our earlier in vitro studies (Hodges et al., 2008). To test if mRNA localization is more efficient when two motors are coupled together, we compared transport by a constitutive dimer of Myo4p/She3p with a constitutive monomer. Correct ASH1 mRNA localization was achieved equally well with both constructs. This may reflect the fact that many mRNAs and thus many motors are part of the translocation complex. Our results show that the most important feature for correct localization is the retention of coupling between all the members of the complex (Myo4p- She3p-She2p-ASH1 mRNA), which is aided by She3p being a tightly bound subunit of Myo4p.