Investigating The Association Of The Myosin Motor Myo2P With Its Secretory Cargo
Cell polarity involves transport of specific membranes and macromolecules at the right time to the right place. In budding yeast, growth is highly asymmetric and requires a constant delivery of secretory cargo to growth sites. This polarized growth depends on an interplay between the cytoskeletal machinery responsible of directed transport and the secretory systems ensuring proper generation and fusion of membrane carriers. Almost all membrane movements are dependent on the myosin-V motor Myo2p, but how Myo2p recognizes and associates with secretory membranes, its only essential cargo, is currently unknown. Here I identify two myo2 alleles that are sensitive to the levels of Golgi PI4P and of the Rab GTPases Sec4p and Ypt31p. I show that PI4P is enriched in the late secretory compartments and is critical for their association with, and transport by, Myo2p. Similarly, I found a direct interaction between the Myo2p tail and the Rabs Ypt31/32p and Sec4p, disruption of which correlates with uncoupling of the secretory cargo from Myo2p. Although the Myo2p tail failed to bind PI4P directly in our assays, enhancing Myo2p's association with PI4P by fusing it to a PH domain specific for this lipid rescues the PI4P-sensitive myo2 alleles as well as a Rab-binding deficient myo2 mutant. I also present evidence that additional proteins can modulate the interaction between Myo2p and the Rabs, supporting a model where a multicomponent complex makes up the secretory vesicle receptor. Taken together, the data presented here support a model where the Rab GTPase Sec4p, and to a lesser extent Ypt31/32p, and the phosphoinositide PI4P facilitates the recruitment of Myo2p to secretory membranes, resulting in their polarized delivery to sites of growth.
myosin motors; phosphoinositides; myo2; sec4; yeast
Bretscher, Anthony Paul
Alani, Eric E; Henry, Susan A.
Molecular and Cell Biology
Ph. D., Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis