STUDIES OF THE NUCLEUS/VACUOLE JUNCTION IN S CEREVISIAE

酿酒酵母细胞核/液泡连接的研究

• 批准号: 7355024
• 负责人: DAVID S GOLDFARB
• 金额: $ 0.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355024
• 关键词: nuclear membrane; tomography; yeasts

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nonessential portions of the yeast nucleus are targeted to the vacuole and degraded by ¿piecemeal microautophy of the nucleus¿ (PMN) (Roberts et al., 2003; Goldfarb, 2003). During PMN, teardrop-like nuclear blebs are engulfed by invaginations of the vacuole membrane, pinched into the vacuole lumen, and degraded by acid hydrolases. PMN occurs in the context of nucleus-vacuole (NV) junctions, which are Velcro-like patches formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p on the outer nuclear membrane (Kvam and Goldfarb, 2004, Kvam et al., 2005). We are applying both standard electron tomography of high pressure frozen, freeze-substituted material and as cryo-electron tomography of frozen hydrated cryosections to study the NV junction in wild-type and mutant strains, including cells expressing the N-terminal truncation of Nvj1p and strains with an expanded N-terminal domain. These studies should provide a unique and informative approach for a detailed structural analysis of this junction and should afford the resolution necessary to identify and characterize putative cross-bridges between membranes. Our initial tomographic studies used yeast that had been high pressure frozen, freeze-substituted and embedded in plastic. These preparations showed that the separation between the inner and outer nuclear envelope is less at the NV junctions than elsewhere, and that the spacing expands in regions away from the NV junction, supporting the hypothesis that Nvj1p may span the perinuclear lumen and connect the inner and outer nuclear membranes. We have also begun to image samples in the frozen-hydrated state by cutting cryosections of yeast that have been high pressure frozen. Cryo-electron tomography of the sections is being preformed to analyze the NV junction in a state that is close to its native condition. Preliminary work shows promising results from profiles of NV junctions in cryosections of wild-type yeast. This emerging technology has the advantage of imaging the NV junctions that are fully hydrated, unfixed and unstained. Combining electron tomography with a genetic approach should yield a better understanding of the NV junction in yeast in particular, and contribute to our understanding of the proteins that connect membrane systems in general.

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。酵母核的非必需部分靶向液泡，并被核的零碎微量萎缩降解（PMN）（Roberts等，2003； Goldfarb，2003）。在PMN期间，泪珠状的核泡被吸尘膜的凹陷所吞没，被捏入真空腔中，并被酸水解酶降解。 PMN发生在核能（NV）连接的背景下，这些连接是通过Velcro样斑块通过真空膜上VAC8P之间的特定相互作用而形成的，外部核膜上（KVAM）和Goldfarb，2004年，2004年，KVAM等人，KVAM等人，2005年）。我们正在应用高压冷冻，冷冻溶剂材料的标准电子断层扫描，也应用了冷冻水合冷冻切除术的冷冻电子层析成像，以研究野生型和突变菌株中的NV连接，包括表达NVJ1P的N-末端截断的细胞以及NVJ1P的N-末端截断，并具有扩展的N-Terminal terminal terminal terminal distrain disminal disminal disminal disminal disminal disminal domain。这些研究应提供对该结的详细结构分析的独特和信息性的方法，并应提供确定和表征机制之间假定的跨桥所必需的分辨率。我们的最初断层扫描研究使用了高压冷冻，冷冻取代并嵌入塑料中的酵母。这些制剂表明，内部和外部核包膜在NV连接处的分离少于其他地方，并且间距在远离NV连接的区域扩展，这支持了NVJ1P可能跨越核周流体并连接内部和外核膜的假设。我们还开始通过切割已经冷冻的酵母的冷冻切片来对样品进行成像。该部分的冷冻电子层析成像已被预先形成，以分析接近其天然状况的状态的NV连接。初步工作表明，野生型酵母冷冻切片中NV连接的概况有望结果。这项新兴技术具有成像完全水合，未固定和未染色的NV连接。将电子断层扫描与通用方法相结合，特别是对酵母中NV连接的理解，并有助于我们理解一般连接膜系统的蛋白质。