Env7 as a Conserved Member of a Novel Kinase Cascade Regulating Membrane Fusion

 描述（由申请人提供）：虽然膜融合/裂变所涉及的分子机器已经被剖析，但融合/裂变动力学的调节仍然知之甚少。我们发现 ENV7 是一种参与液泡事件的新基因，并将其确定为 STK16 相关激酶的直系同源物。我们已经证明，Env7 蛋白是一种棕榈酰化液泡膜激酶，在出芽和高渗应激期间负调节液泡膜融合。此外，我们已经确定 Env7 体内磷酸化依赖于 YCK3，YCK3 是编码另一种液泡膜激酶的基因，在融合调节中具有非冗余功能。这两个基因的缺失会导致严重的形态缺陷和生长受损。因此，Env7 介导的膜通量调节似乎是影响整体细胞适应性的新型激酶级联的一部分。根据我们迄今为止的发现，我们提出了一个调节 Env7 功能的模型，并假设 1) Env7 介导的液泡融合/裂变是通过 Yck3 磷酸化来调节的，2) Env7 底物包括液泡融合/裂变机制的成分。在这里，我们建议通过系统的多方面方法测试所提出模型的各个方面，包括活细胞成像、生化分析、体内和体外融合测定以及全局蛋白质组学研究。如果成功，该项目将把 Env7 建立为参与膜融合/裂变动力学调节的新型激酶信号级联中的节点。这将是细胞器生物发生、膜动力学和信号传导方面的一次飞跃。拟议的实验也代表了对任何 STK16 相关激酶的调节和细胞底物的首次研究。该 SCORE 提案的内在目标是在膜融合和激酶级联的高度活跃领域内发展 PI 的深度和研究生产力，同时在综合性西班牙服务研究所内继续对本科生和硕士生进行富有成效的研究培训。溶酶体缺陷与溶酶体贮积病和神经变性有关。特别是溶酶体融合动力学的缺陷最近已成为与溶酶体贮积病和阿尔茨海默氏病相关的病理学中可能的潜在机制。

 DESCRIPTION (provided by applicant): While molecular machineries involved in membrane fusion/fission have been dissected, regulation of fusion/fission dynamics remains poorly understood. We uncovered ENV7 as a novel gene involved in vacuolar events and have established it as an ortholog of STK16-related kinases. We have shown that Env7 protein is a palmitoylated vacuolar membrane kinase that negatively regulates vacuolar membrane fusion during budding and hyperosmotic stress. Additionally, we have established that Env7 phosphorylation in vivo is dependent on YCK3, a gene encoding another vacuole membrane kinase with a non-redundant function in fusion regulation. Deletion of both genes results in severe morphological defects and compromised growth. Thus, Env7-mediated membrane flux regulation appears to be part of a novel kinase cascade affecting global cell fitness. Based on our findings to date, we propose a model for regulation of Env7 function and hypothesize that 1) Env7-mediated vacuole fusion/fission is regulated through its phosphorylation by Yck3 and 2) Env7 substrate(s) include component(s) of the vacuolar fusion/fission machinery. Here, we propose to test various aspects of the proposed model through a systematic multifaceted approach that includes live cell imaging, biochemical analyses, in vivo and in vitro fusion assays, and global proteomic studies. If successful, this project will establish Env7 as a node in a novel kinase signaling cascade involved in regulation of membrane fusion/fission dynamics. This will be a leap forward in organelle biogenesis, membrane dynamics, and signaling. The proposed experiments also represent the first studies on regulation and cellular substrates of any STK16-related kinase. Inherent in this SCORE proposal, is the aim to develop the depth and research productivity of the PI within the highly active fields of membrane fusion and kinase cascades, while continuing the productive research training of undergraduate and Master's students within a comprehensive Hispanic Serving Institute. Lysosomal defects have been associated with Lysosomal Storage Diseases and neurodegeneration. Defects specifically in lysosomal fusion dynamics have recently emerged as the possible underlying mechanism in pathologies associated with both Lysosomal Storage Diseases and Alzheimer's disease.