title: A point mutation in the cargo-binding domain of myosin V affects its interaction with multiple cargoes.
abstract: Class V myosins move diverse intracellular cargoes, which attach via interaction of cargo-specific proteins to the myosin V globular tail. The globular tail of the yeast myosin V, Myo2p, contains two structural and functional subdomains. Subdomain I binds to the vacuole-specific protein, Vac17p, while subdomain II likely binds to an as yet unidentified secretory vesicle-specific protein. All functions of Myo2p require the tight association of subdomains I and II, which suggests that binding of a cargo to one subdomain may inhibit cargo-binding to a second subdomain. Thus, two types of mutations are predicted to specifically affect a subset of Myo2p cargoes: first are mutations within a cargo-specific binding region; second are mutations that mimic the inhibited conformation of one of the subdomains. Here we analyze a point mutation in subdomain I, myo2-2(G1248D), which is likely to be this latter type of mutation. myo2-2 has no effect on secretory vesicle movement. The secretory vesicle binding site is in subdomain II. However, myo2-2 is impaired in several Myo2p-related functions. While subdomains I and II of myo2-2p tightly associate, there are measurable differences in the conformation of its globular tail. Based solely on the ability to restore vacuole inheritance, a set of intragenic suppressors of myo2-2 were identified. All suppressor mutations reside in subdomain I. Moreover, subdomain I and II interactions occurred in all suppressors, demonstrating the importance of subdomain I and II association for Myo2p function. Furthermore, 3 of the 10 suppressors globally restored all tested defects in myo2-2. This large proportion of global suppressors strongly suggests that myo2-2(G1248) causes a conformational change in subdomain I that simultaneously affects multiple cargoes.
authors.affiliation: Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: Natasha
authors.initials: N
authors.lastname: Pashkova
authors.forename: Natalie L
authors.initials: NL
authors.lastname: Catlett
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Novak
authors.forename: Lois S
authors.initials: LS
authors.lastname: Weisman
mesh_terms: D001665:Binding Sites; D016153:Genes, Suppressor; D018995:Myosin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D024701:Myosin Type V / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D013489:Suppression, Genetic; D020798:Two-Hybrid System Techniques; D014617:Vacuoles
chemical_list: C077512:MYO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D024701:Myosin Type V; D018995:Myosin Heavy Chains