Genetic Analysis of Complex Membrane Protein Insertion

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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. *** ??