MECHANISMS OF ORGANELLE BIOGENESIS AT THE ENDOPLASMIC RETICULUM SUBDOMAINS

项目总结 细胞内膜结合细胞器是所有真核细胞的标志。了解细胞如何 产生不同的细胞器仍然是细胞生物学的中心问题之一。一些细胞器，如 内质网(ER)和线粒体是自我生成的，而其他细胞器可以 产生了从头开始。内质网在细胞器的生物发生中起着核心作用。即使急诊室是一个单一的 从外核膜延伸到细胞外围的连续膜，有 ER膜上的离散区域称为ER亚域。新生过氧化体和脂滴(LDS) 在专门的ER子域中形成。值得注意的是，人们对这些ER亚域及其作用知之甚少 在调节细胞器的生物发生方面。我们研究的目标是确定过氧化物酶的作用机制。 和LD的生物发生通过详细表征离散的ER亚域酿酒酵母和 哺乳动物细胞培养。在此之前，我们发现了一个网状ER膜微管蛋白家族， 酵母中的Pex30和含有跨膜蛋白MCTP1和MCTP2的多个C2结构域 高等真核生物。我们证明了Pex30和MCTP都定位于离散的ER亚域 在那里形成了新生的过氧体前囊泡和LDS。基于这些发现，我们建议 使用无偏倚以及 基于候选人的方法。然后我们将测试调节候选蛋白质功能的效果 以及脂质对过氧化体和LD的形成、丰度、形态和分布的影响。调查 来自这些ER亚区的过氧体和LDS生物发生的机制细节不仅是 对于理解细胞生物学的基本原理很重要，但也具有重要的医学意义。几个 包括Zellweger综合征在内的威胁生命的神经系统疾病与过氧化物体缺陷相关 而由LD缺陷引起的代谢紊乱，如2型糖尿病和脂肪肝，则没有 解药。确定细胞器生物发生的机制将对理解 这些疾病的病理生理学，并为我们提供潜在的治疗靶点的提示。

PROJECT SUMMARY Intracellular membrane-bound organelles are a hallmark of all eukaryotic cells. Understanding how cells generate different organelles remains one of the central problems in cell biology. Some organelles, like the endoplasmic reticulum (ER) and mitochondria, are self-generating whereas other organelles can be generated de novo. The ER plays a central role in organelle biogenesis. Even though the ER is a single continuous membrane that extends from the outer nuclear envelope into the periphery of the cell, there are discrete regions in the ER membrane called ER subdomains. Nascent peroxisomes and lipid droplets (LDs) form at specialized ER subdomains. Remarkably, little is known about these ER subdomains and their role in regulating organelle biogenesis. The goal of our research is to determine the mechanisms of peroxisome and LD biogenesis by detailed characterization of the discrete ER subdomains using S. cerevisiae and mammalian cell culture. Previously, we identified a family of reticulon-like ER membrane tubulating proteins, Pex30 in yeasts and multiple C2 domains containing transmembrane proteins, MCTP1 and MCTP2, in higher eukaryotes. We demonstrated that both Pex30 and MCTPs are localized at discrete ER subdomains where nascent pre-peroxisomal vesicles and LDs are formed. Based on these findings, we proposed to identify the proteins and lipids enriched at the specialized ER subdomains using unbiased as well as candidate-based approaches. We will then test the effects of modulating the functions of candidate proteins and lipids on the formation, abundance, morphology, and distribution of peroxisomes and LDs. Investigating the mechanistic details of peroxisomes and LDs biogenesis from these ER subdomains is not only important for understanding basic principles of cell biology but also has critical medical implications. Several life-threatening neurological disorders including Zellweger syndrome associated with peroxisomal defects and metabolic disorders such as type 2 diabetes and fatty liver disease caused due to LD defects have no cure. Determining the mechanisms of organelle biogenesis will have implications in understanding the pathophysiology of these disorders and provide us hints for potential therapeutic targets.