title: Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen.
abstract: Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.
authors.affiliation: Department of Biochemistry,University of Puerto Rico, San Juan, Puerto Rico 00936-5067.
authors.forename: Ednalise
authors.initials: E
authors.lastname: Santiago
authors.affiliation: Department of Biochemistry,University of Puerto Rico, San Juan, Puerto Rico 00936-5067 Center for Molecular Sciences and Research, University of Puerto Rico, San Juan, Puerto Rico 00926.
authors.forename: Pearl
authors.initials: P
authors.lastname: Akamine
authors.affiliation: Department of Biochemistry, Department of Molecular Genetics, Donnelly Centre, University of Toronto, Ontario M5S 3E1, Canada.
authors.forename: Jamie
authors.initials: J
authors.lastname: Snider
authors.affiliation: Department of Biochemistry, Department of Molecular Genetics, Donnelly Centre, University of Toronto, Ontario M5S 3E1, Canada.
authors.forename: Victoria
authors.initials: V
authors.lastname: Wong
authors.affiliation: Department of Biochemistry, University of Regina, Saskatchewan, Canada.
authors.forename: Matthew
authors.initials: M
authors.lastname: Jessulat
authors.affiliation: Department of Biochemistry, University of Regina, Saskatchewan, Canada.
authors.forename: Viktor
authors.initials: V
authors.lastname: Deineko
authors.affiliation: Department of Biochemistry, College of Medicine, University of Saskatchewan, Canada.
authors.forename: Alla
authors.initials: A
authors.lastname: Gagarinova
authors.affiliation: Department of Biochemistry, University of Regina, Saskatchewan, Canada.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Aoki
authors.affiliation: Department of Biochemistry, University of Regina, Saskatchewan, Canada.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Minic
authors.affiliation: Department of Biochemistry, University of Regina, Saskatchewan, Canada.
authors.forename: Sadhna
authors.initials: S
authors.lastname: Phanse
authors.affiliation: Department of Biochemistry,University of Puerto Rico, San Juan, Puerto Rico 00936-5067.
authors.forename: Andrea
authors.initials: A
authors.lastname: San Antonio
authors.affiliation: School of Medicine, Universidad Central del Caribe, Bayamon, Puerto Rico 00960-6032.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Cubano
authors.affiliation: Department of Biology, University of Kentucky, Lexington, Kentucky 40506.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Rymond
authors.affiliation: Department of Biochemistry, University of Regina, Saskatchewan, Canada.
authors.forename: Mohan
authors.initials: M
authors.lastname: Babu
authors.affiliation: Department of Biochemistry, Department of Molecular Genetics, Donnelly Centre, University of Toronto, Ontario M5S 3E1, Canada.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
authors.affiliation: Department of Biochemistry,University of Puerto Rico, San Juan, Puerto Rico 00936-5067 jose.rodriguez123@upr.edu.
authors.forename: Jose R
authors.initials: JR
authors.lastname: Rodriguez-Medina
mesh_terms: D047468:Immunoprecipitation / Q000379:methods; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D024462:Myosin Type II
keywords: Myo1p; cytokinesis; interactome; proteomics; yeast