Estravís Miguel, Rincón Sergio Antonio, Portales Elvira, Pérez Pilar, Santos Beatriz
Instituto de Biología Funcional y Genómica (IBFG), Consejo Superior de Investigaciones Científicas, University of Salamanca, 37007 Salamanca, Spain.
Present address: Institut Curie, Centre de Recherche, PSL Research University, CNRS UMR144, F-75248 Paris, France.
Microbiology (Reading). 2017 Aug;163(8):1156-1166. doi: 10.1099/mic.0.000503.
Rho GTPases control polarized cell growth and are well-known regulators of exocytic and endocytic processes. Cdc42 is an essential GTPase, conserved from yeast to humans, that is critical for cell polarization. Cdc42 is negatively regulated by the GTPase-activating proteins (GAPs) and the GDP dissociation inhibitors (GDIs), and positively regulated by guanine nucleotide exchange factors (GEFs). Cdc42 GTPase can be found in a GTP- or GDP-bound state, which determines the ability to bind downstream effector proteins and activate signalling pathways. Only GTP-bound Cdc42 is active. In this study we have analysed the localization of the different nucleotide-bound states of Cdc42 in : the wild-type Cdc42 protein that cycles between an active and inactive form, the Cdc42G12V form that is permanently bound to GTP and the Cdc42T17N form that is constitutively inactive. Our results indicate that Cdc42 localizes to several membrane compartments in the cell and this localization is mediated by its C-terminal prenylation. Constitutively active Cdc42 localizes mainly to the plasma membrane and concentrates at the growing tips where it is considerably less dynamic than wild-type or GDP-bound Cdc42. Additionally we show that the activation state of Cdc42 also participates in the regulation of its protein levels mediated by endocytosis and by the exocyst complex.
Rho GTP酶控制细胞的极性生长，是胞吐和胞吞过程中众所周知的调节因子。Cdc42是一种必需的GTP酶，从酵母到人类都保守存在，对细胞极化至关重要。Cdc42受到GTP酶激活蛋白（GAP）和GDP解离抑制剂（GDI）的负调控，并受到鸟嘌呤核苷酸交换因子（GEF）的正调控。Cdc42 GTP酶可以处于结合GTP或GDP的状态，这决定了其结合下游效应蛋白并激活信号通路的能力。只有结合GTP的Cdc42才具有活性。在本研究中，我们分析了Cdc42不同核苷酸结合状态在以下方面的定位：在活性和非活性形式之间循环的野生型Cdc42蛋白、永久结合GTP的Cdc42G12V形式以及组成型无活性的Cdc42T17N形式。我们的结果表明，Cdc42定位于细胞内的几个膜区室，这种定位由其C末端异戊二烯化介导。组成型活性Cdc42主要定位于质膜，并集中在生长尖端，在那里它的动态性比野生型或结合GDP的Cdc42低得多。此外，我们还表明，Cdc42的激活状态也参与了由内吞作用和外被复合体介导的其蛋白质水平的调节。
