Genetic Analysis of Complex Membrane Protein Insertion

9506989 Manoil, Colin C.该项目研究大肠杆菌渗透酶是如何在膜中组装的，以及突变变化是如何破坏这一过程的。1. 我们的目的是确定lac渗透酶的内部极性结构域（P6）跨膜的易位是否由邻近该结构域的每个跨膜序列冗余决定。为了做到这一点，我们将研究当c端跨膜序列（TM12）存在时，失活更多n端跨膜序列（TM11）的单个输出功能的突变是否被抑制。2. 我们将鉴定一大批失活紫胶渗透酶的错义突变。许多变化应该会改变膜插入蛋白的折叠，突变集应该为这一过程的系统分子遗传学分析提供基础。将检测突变蛋白的细胞稳定性、膜拓扑结构和正常折叠能力。3. 我们正在鉴定紫胶渗透酶TM12突变导致该蛋白对细胞具有高度毒性。我们将通过分析对有毒蛋白的敏感性增加或减少的突变体来研究毒性的机制。4. 我们将从球形红杆菌中鉴定出改变光合反应L亚基最c端跨膜序列（TM5）的失活突变和中性突变。我们将描述面对蛋白质其他部分的跨段侧与面对脂质的侧对不同取代类型的相对敏感性。这一分析将为解释未知结构的膜蛋白的类似研究提供指导。此外，我们将确定第二位点可逆分析是否可以识别与TM5相邻的跨膜序列残基。如果是这样的话，对还原物的研究可以为其他结构未知的膜蛋白（如紫胶渗透酶）的折叠结构元素的鉴定提供一种通用的方法。5. 我们正在构建一个反式子衍生物，它将融合所有的lac渗透酶，除了它的最后一个跨膜序列（TM12）到靶基因产物。对LacY+转座子插入的分析应该能够确定哪些序列可以在功能上替代TM12，并可能为鉴定紫胶渗透酶相关蛋白的基因提供一种方法。膜蛋白在多种细胞功能中起着重要作用，例如光合作用。这项研究将使人们了解蛋白质是如何插入细胞膜的，以及它们在细胞膜上是如何起作用的。利用这些信息，可能会产生具有所需结构和功能的新膜蛋白。* * * ? ?

9506989 Manoil, Colin C. The project investigates how Escherichia coli lac permease is assembled in the membrane and how mutational changes disrupt the process. 1. We aim to determine whether the translocation of an internal polar domain (P6) of lac permease across the membrane is redundantly determined by each of the transmembrane sequences adjacent to the domain. To do this, we will examine whether mutations inactivating the individual export function of the more N-terminal transmembrane sequence (TM11 ) are suppressed when the C-terminal transmembrane sequence (TM12) is present. 2. We will identify a large set of missense mutations inactivating lac permease. Many of the changes should alter folding of the membrane-inserted protein, and the mutant set should provide a foundation for a systematic molecular genetic analysis of this process. Mutant proteins will be assayed for cellular stability, membrane topology and ability to fold normally. 3. We are identifying lac permease TM12 mutations that cause the protein to be highly toxic to cells. We will investigate the mechanism of toxicity by analyzing mutants that show increased or decreased sensitivity to the toxic proteins. 4. We will identify inactivating and neutral mutations altering the most C-terminal transmembrane sequence (TM5) of the L subunit of the photosynthetic reaction from Rhodobacter sphaeroides. We will characterize the relative sensitivity to different substitution types of the side of the spanning segment facing other parts of the protein versus the side facing lipid. This analysis will serve as a guide for interpreting analogous studies of membrane proteins of unknown structure. In addition, we will determine whether second site revertant analysis can identify residues in transmembrane sequences adjacent to TM5. If so, the study of revertants may provide a general method for identifying elements of the folded structure of other membrane proteins of unknown structure, such as lac permease. 5. We are constructing a trans poson derivative that will function to fuse all of lac permease except its last transmembrane sequence (TM12) to target gene products. Analysis of LacY+ transposon insertions should make it possible to determine what sequences can functionally substitute for TM12 and may provide a way to identify genes for proteins related to lac permease. %%% Membrane proteins play fundamental roles in a variety of cellular functions, such as photosynthesis. This research will lead to an understanding of how proteins are inserted into membranes and how they function there. New membrane proteins with desired structures and functions may be able to be created using this information. *** ??