Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen

Nonmuscle myosin type II ([Myo1p][1]) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae . Loss of [Myo1p][1] activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The [Myo1p][1] tail-only domain was previously reported to have functional activity equivalent to the full-length [Myo1p][1] whereas the head-only domain did not. Since [Myo1p][1] 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][1]-interacting proteins may shed light on the other functions of the [Myo1p][1] tail domain. To identify novel [Myo1p][1]-interacting proteins, and determine if [Myo1p][1] 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][1] 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][1]-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel [Myo1p][1]-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation ([Ape2][2], [Bzz1][3], [Fba1][4], [Pdi1][5], [Rpl5][6], [Tah11][7], and [Trx2][8]) or mass spectrometry (AP-MS) ([Abp1][9]). The novel [Myo1p][1]-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][3] and the actin-binding protein [Abp1][9] represent likely [Myo1p][1] interactions during cytokinesis. [1]: http://www.yeastgenome.org/locus/S000001065/overview [2]: http://www.yeastgenome.org/locus/S000001640/overview [3]: http://www.yeastgenome.org/locus/S000001156/overview [4]: http://www.yeastgenome.org/locus/S000001543/overview [5]: http://www.yeastgenome.org/locus/S000000548/overview [6]: http://www.yeastgenome.org/locus/S000006052/overview [7]: http://www.yeastgenome.org/locus/S000003807/overview [8]: http://www.yeastgenome.org/locus/S000003441/overview [9]: http://www.yeastgenome.org/locus/S000000684/overview