Shannon K B, Li R
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Biol Cell. 1999 Feb;10(2):283-96. doi: 10.1091/mbc.10.2.283.
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 COOH-terminal 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 calcium-dependent 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.
出芽酵母中类IQGAP蛋白Cyk1p/Iqg1p在后期定位于母细胞与芽的交界处，并且已证明它是胞质分裂完成所必需的。在本研究中，对表达绿色荧光蛋白标记的Cyk1p/Iqg1p的细胞进行视频显微镜分析表明，Cyk1p/Iqg1p是胞质分裂期间收缩环的一个动态组成部分。此外，在没有Cyk1p/Iqg1p的情况下，肌球蛋白II在有丝分裂末期无法经历类似收缩的大小变化。为了了解Cyk1p/Iqg1p在肌动球蛋白环组装和动态变化中的机制作用，我们研究了Cyk1p/Iqg1p与IQGAPs共有的结构域的作用。含有钙调蛋白同源结构域的氨基末端部分与肌动蛋白丝结合，并且是肌动蛋白丝组装到环上所必需的。这一结果支持了Cyk1p/Iqg1p在F-肌动蛋白募集过程中起直接作用的假设。缺失含有八个IQ基序的结构域会消除Cyk1p/Iqg1p在芽颈处的定位，这表明Cyk1p/Iqg1p可能通过与一种类钙调蛋白的蛋白质相互作用而定位。有趣的是，缺失COOH末端的GTP酶激活蛋白相关结构域并不影响Cyk1p/Iqg1p的定位或肌动蛋白募集到环上，但会阻止肌动球蛋白环的收缩。体外结合实验表明，Cyk1p/Iqg1p以钙依赖的方式与钙调蛋白Cmd1p结合，并与Tem1p结合，Tem1p是一种先前发现的对后期完成所必需的小GTP结合蛋白。这些结果证明了Cyk1p/Iqg1p在调节肌动球蛋白环组装和胞质分裂的各个步骤中的关键功能。
