Membrane Protein Topology in Yeast

酵母中的膜蛋白拓扑结构

• 批准号: 9219974
• 负责人: Donald Tipper
• 金额: $ 27万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-03-15 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9219974&HistoricalAwards=false
• 关键词: Membrane; Protein; Topology; Yeast

This laboratory has developed a procedure for using beta- lactamase (beta-1a) for analysis of the topology of transmembrane proteins expressed in yeast. A C-terminal beta-1a reporter is fused to N-terminal fragments of target proteins via the P fragment of Killer preprotoxin. In fusions in which this P-beta-1a reporter is lumenal, processing of P in a late Golgi compartment by the Kex2 protease results in secretion of beta-1a. Analysis of Kex2- dependent beta-1a secretion, with and without an intervening signal sequence S, provides positive identification of both cytoplasmic (internal) and "periplasmic" (external) fusion sites. A model for the Ste2 receptor was produced that agreed with predictions. Corroborative data should result from analysis of the size, N- glycosylation pattern and protease sensitivity of the intact fusion proteins expressed in a kex2 mutant, or expressed in vitro. The technique is being used to analyze the topology of Pma1, the yeast plasma membrane proton ATPase, a model for the family of P-type ATPase ion pumps found in all cell types. Any ambiguities in deduced topology will be resolved using a sandwich technique, using either beta-1a or P-beta-1a as the inserted reporter. Analyses of Pma1 function by site-directed mutagenesis have identified many residues important to the conserved cytoplasmic kinase and phosphatase domains, but have had very limited success in identifying residues critical to proton channel function. Our preliminary Pma1 topology identifies new candidate residues; if the topology is confirmed, the phenotypes of mutants in these residues should clarify the ion pump mechanism. %%% A thorough understanding of protein function (and the ability to use such an understanding to engineer proteins with desired properties) depends on knowledge of three-dimensional structure. Unfortunately, the very properties of integral membrane proteins that allow their transmembrane segments to interact with the lipid biolayer make it impossible to apply the usual approaches for purification and crystallization that are normally employed for determining 3-D protein structures. Thus, structural information on membrane-spanning proteins is particularly difficult to obtain. This is frustrating, because some of the most interesting proteins from a functional point of view are membrane-spanning proteins (receptors, pumps, channels, etc.). This project offers a clever approach to the dilemma, which involves topological analysis of membrane proteins through genetic engineering. The idea is to construct chimeric mutants of the membrane protein of interest in which the introduced piece has a known proteolytic cleavage site that only gets cleaved if it is facing the lumen of the Golgi. By determining which constructs undergo cleavage, one can deduce the locations of the transmembrane regions of the protein, and from that deduce 3-D structure. Although the project focuses on a specific transmembrane proton pump, the approach can be generalized to a wide variety of biologically important proteins. Many downstream biotechnological applications of both the approach and the structural information obtained can easily be envisioned.

这个实验室已经开发出一种程序， 用于分析跨膜拓扑结构的内酰胺酶（β-1a） 在酵母中表达的蛋白质。 一种C-末端β-1a报告基因， 通过P片段与靶蛋白的N-末端片段融合 杀手前原毒素 在这种融合中， 是内腔的，由Kex 2在晚期高尔基体室中处理P 蛋白酶导致β-1a的分泌。 分析Kex 2- 依赖性β-1a分泌，有和没有干预信号 序列S提供了两种细胞质的阳性鉴定， （内部）和“周质”（外部）融合位点。 的典范 Ste 2受体的产生与预测一致。 确证性数据应来自对尺寸的分析，N- 完整融合体的糖基化模式和蛋白酶敏感性 在kex 2突变体中表达或在体外表达的蛋白质。 的 技术被用来分析Pma 1的拓扑结构， 质膜质子ATP酶，一个模型的家庭的P型 ATP酶离子泵存在于所有细胞类型中。 中的任何含糊不清 推导出的拓扑结构将使用三明治技术解决，使用 β-1a或P-β-1a作为插入的报告基因。 分析 Pma 1功能的定点突变已经确定了许多 对保守的细胞质激酶重要的残基， 磷酸酶结构域，但在 鉴定对质子通道功能至关重要的残基。 我们 初步Pma 1拓扑结构鉴定新的候选残基;如果 拓扑结构得到证实，这些残基中突变体的表型 应该澄清离子泵的机制。 %%% 对蛋白质功能的透彻理解（以及 利用这样的理解来设计蛋白质， 性质）取决于三维结构的知识。 不幸的是，膜蛋白的特性 使它们的跨膜片段与脂质相互作用 生物层使其不可能应用通常的方法， 纯化和结晶通常用于 确定蛋白质的三维结构 因此，结构信息 对跨膜蛋白质的作用是特别难以获得的。 这是令人沮丧的，因为一些最有趣的蛋白质 从功能的角度来看是跨膜蛋白 （受体、泵、通道等）。 这个项目提供了一个聪明的 方法的困境，其中涉及拓扑分析， 膜蛋白基因工程。 这个想法是 构建目的膜蛋白的嵌合突变体， 所述引入的片段具有已知的蛋白水解切割位点 只有面对高尔基体的内腔才会被切开。 通过 确定哪些构建体经历切割，可以推断出 蛋白质跨膜区的位置，以及 来推断三维结构。 虽然该项目的重点是 特异性跨膜质子泵，该方法可以推广 一系列重要的生物蛋白质。 许多 下游生物技术应用的方法和 所获得的结构信息可以容易地被设想。