The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast

Abstract

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOHterminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calciumdependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.

Mechanisms of cytokinesis in budding yeast

“…The AMR is assembled normally but fails to constrict in cells carrying an iqg1 allele that encodes a truncated protein lacking the GRD (GAP-related domain) [Shannon and Li, 1999]. In addition, Tem1 in either GDP-or GTP-bound state interacts directly with the GRD of Iqg1 [Shannon and Li, 1999]. How the Tem1-GRD interaction controls AMR constriction remains to be investigated.…”

“…Also required for AMR-independent cytokinesis [Epp and Chant, 1997;Shannon and Li, 1999;Boyne et al, 2000;Ko et al, 2007;Tully et al, 2009;Fang et al, 2010] Bni1 Formin Plays a major role in actin ring assembly [Vallen et al, 2000;Pruyne et al, 2004;Buttery et al, 2007] …”

“…Catalytic subunit of chitin synthase III Required for the synthesis of chitin in the ''bud scar'' and the cell wall and may also play a role in SS formation [Shaw et al, 1991;Chuang and Schekman, 1996 [Kovacech et al, 1996] Chs1 Chitin synthase I Involved in septum repair at low pH after cell separation [Cabib et al, 1989;Cabib et al, 1992] Coordination of actomyosin ring and primary septum formation Septins GTP-binding and filament-forming proteins Required for AMR assembly and PS formation [Hartwell, 1971;Longtine et al, 1996 (p 297);Mino et al, 1998;Takizawa et al, 2000;Roh et al, 2002;Dobbelaere and Barral, 2004] Mlc1 ELC Required for AMR assembly and PS formation [Stevens and Davis, 1998;Boyne et al, 2000;Shannon and Li, 2000;Wagner et al, 2002;Luo et al, 2004;Fang et al, 2010] Iqg1 IQGAP Required for AMR assembly and PS formation [Epp and Chant, 1997;Shannon and Li, 1999;Boyne et al, 2000;Korinek et al, 2000;Ko et al, 2007;Tully et al, 2009;Fang et al, 2010] (Continued) n 720…”

The IQGAP1 Protein Is a Calmodulin-regulated Barbed End Capper of Actin Filaments

“…It is possible that binding of other ligands or covalent modifications of IQGAP1 like phosphorylation of Ser-1443 by PKC⑀ (56) up-regulate its capping activity. The fact that a mutant of IQGAP1 that lacks the C-terminal region is unable to promote cytokinesis in yeast but does localize to the cytokinetic ring (57), suggests that the barbed end capping activity in the C-terminal region of IQGAP1 supports its function in cytokinesis, whereas the CHD at the N-terminal is sufficient for its localization.…”

Advances in Cytokinesis Research. Cytokinesis in Budding Yeast: The Relationship between Actomyosin Ring Function and Septum Formation.

“…Iqg1p forms a single ring at the bud neck around anaphase and contracts like Myo1p, suggesting that Iqg1p may be a component of the actomyosin ring (Shannon and Li, 1999). Indeed, Iqg1p is required for the actin ring formation (Epp and Chant, 1997;Lippincott and Li, 1998b).…”