Membrane Protein Topology in Yeast

本实验室开发了一种使用β-内酰胺酶(β-1a)来分析酵母中表达的跨膜蛋白的拓扑结构的方法。通过Killer前原毒素的P片段，将C端的β-1a报告基因融合到靶蛋白的N端片段上。在这种P-β-1a报告者为腔的融合中，在高尔基体晚期的隔室中，Kex2蛋白酶处理P会导致β-1a的分泌。对依赖Kex2的β-1a分泌的分析，在有和没有S干预信号序列的情况下，提供了细胞质(内部)和“周质”(外部)融合位点的阳性鉴定。一个Ste2受体的模型被制作出来，与预测相符。对在kex2突变体中表达或在体外表达的完整融合蛋白的大小、N-糖基化模式和蛋白酶敏感性的分析应得到确证数据。这项技术正被用于分析Pma1的拓扑结构，Pma1是酵母质膜质子ATPase的模型，是在所有细胞类型中发现的P型ATPase离子泵家族的模型。将使用夹心技术，使用beta-1a或P-beta-1a作为插入的报告，解决推导出的拓扑中的任何歧义。通过定点突变对Pma1功能的分析已经发现了许多对保守的细胞质激酶和磷酸酶结构域很重要的残基，但在鉴定对质子通道功能至关重要的残基方面取得的成功非常有限。我们初步的Pma1拓扑确定了新的候选残基；如果拓扑得到确认，这些残基中突变的表型应该会阐明离子泵机制。%对蛋白质功能的透彻理解(以及利用这种理解来设计具有所需性质的蛋白质的能力)取决于对三维结构的了解。不幸的是，完整的膜蛋白允许其跨膜片段与脂质生物层相互作用，这使得通常用于确定三维蛋白质结构的纯化和结晶方法不可能应用。因此，关于跨膜蛋白质的结构信息尤其难以获得。这是令人沮丧的，因为从功能的角度来看，一些最有趣的蛋白质是跨膜蛋白质(受体、泵、通道等)。这个项目提供了一种巧妙的方法来解决这一困境，这涉及到通过基因工程对膜蛋白进行拓扑分析。这个想法是构建感兴趣的膜蛋白的嵌合突变体，其中引入的片段具有已知的蛋白水解性切割位置，只有当它面对高尔基体腔时才被切割。通过确定哪些结构经历了切割，人们可以推断出蛋白质跨膜区的位置，并由此推断出三维结构。尽管该项目专注于一种特定的跨膜质子泵，但该方法可以推广到各种具有生物重要性的蛋白质上。可以很容易地预见该方法和所获得的结构信息的许多下游生物技术应用。

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.