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

 描述(由申请人提供)：尽管涉及膜融合/裂变的分子机制已经被剖析，但对融合/裂变动力学的调节仍然知之甚少。我们发现ENV7是一个与空泡事件有关的新基因，并已将其确定为STK16相关激酶的同源基因。我们已经证明，Env7蛋白是一种棕榈酰化的液泡膜蛋白，在萌发和高渗胁迫过程中负向调节液泡膜的融合。此外，我们还证实了Env7在体内的磷酸化依赖于YCK3基因，YCK3编码另一个在融合调节中具有非冗余功能的液泡膜蛋白激酶。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全球细胞适应性的新的激酶级联的一部分。根据我们到目前为止的发现，我们提出了一个调控Env7功能的模型，并假设1)Env7介导的液泡融合/分裂是通过YKK3的磷酸化来调节的；2)Env7底物(S)包括空泡融合/分裂机制的组成部分(S)。在这里，我们建议通过一个系统的多方面的方法来测试所提出的模型的各个方面，其中包括活细胞成像、生化分析、体内和体外融合分析以及全球蛋白质组研究。如果成功，该项目将使Env7成为参与调控膜融合/裂变动力学的新的激酶信号级联中的一个节点。这将是细胞器生物发生、膜动力学和信号传递方面的一次飞跃。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。这项计分建议的内在目的是在高度活跃的膜融合和激酶级联领域内发展PI的深度和研究生产力，同时继续在一个综合性的西班牙裔服务学院内对本科生和硕士学生进行生产性研究培训。溶酶体缺陷与溶酶体储存疾病和神经退行性变有关。溶酶体融合动力学方面的缺陷最近被认为是溶酶体储存性疾病和阿尔茨海默病相关病理的可能潜在机制。

 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.