National Institute of Plant Genome Research, New Delhi, India.
Department of Molecular Biology and Biotechnology, University of Kalyani, West Bengal, India.
mSphere. 2016 Aug 3;1(4). doi: 10.1128/mSphere.00080-16. eCollection 2016 Jul-Aug.
Vesicular dynamics is one of the very important aspects of cellular physiology, an imbalance of which leads to the disorders or diseases in higher eukaryotes. We report the functional characterization of a palmitoylated protein kinase from Candida albicans whose homologue in Saccharomyces cerevisiae has been reported to be involved in negative regulation of membrane fusion and was named Env7. However, the downstream target of this protein remains to be identified. Env7 in C. albicans (CaEnv7) could be isolated from the membrane fraction and localized to vesicular structures associated with the Golgi apparatus. Our work reports Env7 in C. albicans as a new player involved in maintaining the functional dynamics at the trans-Golgi network (TGN) by interacting with two other TGN-resident proteins, namely, Imh1p and Arl1p. Direct interaction could be detected between Env7p and the golgin protein Imh1p. Env7 is itself phosphorylated (Env7p) and phosphorylates Imh1 in vivo. An interaction between Env7 and Imh1 is required for the targeted localization of Imh1. CaEnv7 has a putative palmitoylation site toward both N and C termini. An N-terminal palmitoylation-defective strain retains its ability to phosphorylate Imh1 in vitro. An ENV7 homozygous mutant showed compromised filamentation in solid media and attenuated virulence, whereas an overexpressed strain affected cell wall integrity. Thus, Env7 plays a subtle but important role at the level of multitier regulation that exists at the TGN. IMPORTANCE A multitier regulation exists at the trans-Golgi network in all higher organisms. We report a palmitoylated protein kinase, Env7, that functions at the TGN interface by interacting with two more TGN-resident proteins, namely, Imh1 and Arl1. Palmitoylation seems to be important for the specific localization. This study focuses on the involvement of a ubiquitous protein kinase, whose substrates had not yet been reported from any organism, as an upstream signaling component that modulates the activity of the Imh1-Arl1 complex crucial for maintaining membrane asymmetry. Virulence is significantly diminished in an Env7 mutant. The functioning of this protein in C. albicans seems to be quite different from its nearest homologue in S. cerervisiae, which reflects the evolutionary divergence between these two organisms.
囊泡动力学是细胞生理学的一个非常重要的方面，其失衡会导致高等真核生物的紊乱或疾病。我们报告了一种来自白色念珠菌的棕榈酰化蛋白激酶的功能特征，其在酿酒酵母中的同源物已被报道参与膜融合的负调节，并被命名为Env7。然而，该蛋白的下游靶标仍有待确定。来自白色念珠菌的 Env7（CaEnv7）可以从膜部分中分离出来，并定位于与高尔基体相关的囊泡结构中。我们的工作报告了白色念珠菌中的 Env7 作为一种新的参与者，通过与另外两个驻留在 TGN 的蛋白质，即 Imh1p 和 Arl1p 相互作用，参与维持 TGN 功能动态。可以检测到 Env7p 与高尔基蛋白 Imh1p 之间的直接相互作用。Env7 本身被磷酸化（Env7p）并在体内磷酸化 Imh1。Env7 和 Imh1 之间的相互作用是 Imh1 靶向定位所必需的。CaEnv7 具有朝向 N 和 C 末端的假定棕榈酰化位点。N 端棕榈酰化缺陷株在体外仍保留磷酸化 Imh1 的能力。ENV7 纯合突变体在固体培养基中的丝状形成能力受损，毒力减弱，而过表达株则影响细胞壁完整性。因此，Env7 在 TGN 存在的多层次调节水平上发挥着微妙但重要的作用。
在所有高等生物中，都存在一个多层次的调节网络在 TGN 中。我们报告了一种棕榈酰化蛋白激酶 Env7，它通过与另外两个驻留在 TGN 的蛋白质，即 Imh1 和 Arl1 相互作用，在 TGN 界面发挥作用。棕榈酰化似乎对特定的定位很重要。这项研究集中在一种普遍存在的蛋白激酶上，其底物尚未在任何生物体中报道，作为一种上游信号成分，调节 Imh1-Arl1 复合物的活性，该复合物对于维持膜不对称性至关重要。Env7 突变体的毒力显著降低。这种蛋白在白色念珠菌中的功能似乎与在酿酒酵母中最接近的同源物非常不同，这反映了这两个生物体之间的进化分歧。
