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

 描述（由申请人提供）：虽然已经解剖了参与膜融合/裂变的分子机制，但对融合/裂变动力学的调节仍然知之甚少。我们发现ENV 7是一个参与液泡事件的新基因，并将其确定为STK 16相关激酶的直系同源物。我们已经表明，Env 7蛋白是棕榈酰化的液泡膜激酶，负调节液泡膜融合过程中出芽和高渗胁迫。此外，我们已经确定Env 7在体内的磷酸化依赖于YCK 3，YCK 3是编码另一种在融合调节中具有非冗余功能的液泡膜激酶的基因。这两个基因的缺失导致严重的形态缺陷和受损的生长。因此，Env 7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。基于我们迄今为止的发现，我们提出了一个调控Env 7功能的模型，并假设1）Env 7介导的液泡融合/分裂是通过Yck 3的磷酸化调控的，2）Env 7底物包括液泡融合/分裂机制的组分。在这里，我们建议通过一个系统的多方面的方法，包括活细胞成像，生化分析，在体内和体外融合试验，和全球蛋白质组学研究，以测试所提出的模型的各个方面。如果成功，该项目将建立Env 7作为一个新的激酶信号级联参与调节膜融合/裂变动力学的节点。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STK 16相关激酶的调节和细胞底物的首次研究。在这个SCORE提案中固有的目的是在膜融合和激酶级联的高度活跃的领域内发展PI的深度和研究生产力，同时继续在综合的西班牙裔服务研究所内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经成为与溶酶体贮积病和阿尔茨海默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.