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.
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The research reported in this publication was supported by awards from the National Institute on Minority Health and Health Disparities (NIMHD) of the National Institutes of Health (NIH) to the University of Hawaii (award number U54MD008149), and to the University of Puerto Rico (award numbers G12MD007600 and U54MD007587). Additional support was provided by awards from the National Institute for General Medical Sciences (NIGMS) to the University of Puerto Rico (award numbers R25GM061838 and P20GM103475), and a Title V PPOHA grant from the United States (US) Department of Education to Universidad Central del Caribe (award number P031M105050). This research was also supported by the Canadian Institutes of Health Research grant (MOP: 125952) to M.B. Work in the Stagljar Laboratory is supported by grants from the Ontario Genomics Institute, Canadian Cystic Fibrosis Foundation, Canadian Cancer Society, Pancreatic Cancer Canada, and University Health Network.
Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.115.026609/-/DC1
Santiago, Ednalise; Akamine, Pearl; Snider, Jamie; Wong, Victoria; Jessulat, Matthew; Deineko, Viktor; Gagarinova, Alla; Aoki, Hiroyuki; Minic, Zoran; Phanse, Sadhna; San Antonio, Andrea; Cubano, Luis A; Rymond, Brian C.; Babu, Mohan; Stagljar, Igor; and Rodriguez-Medina, Jose R., "Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen" (2016). Biology Faculty Publications. 106.