This project addresses the problem of how the information contained in the amino acid sequence of a secreted or a membrane protein determines the precise topology of insertion into or through the membrane. This is a fundamental problem in cell biology. Positively charged residues, often found located near the boundaries of transmembrane segments of proteins, appear to be involved in specifying the orientation of membrane proteins. This proposal examines in detail to what extent positively charged residues play a part in this process and the mechanism by which proteins obtain their unique membrane topology in Escherischia coli. The aims of the proposal are: 1, to determine whether inner membrane proteins, like other exported proteins in E. coli, require molecular chaperones for their membrane assembly; 2, to define genetically the parts of the proteins that participate in their membrane assembly; 3, to evaluate the mechanisms of membrane insertion for simple and complex membrane proteins; 4, to establish the general role of positively charged domains in protein orientation; and 5, to determine how positively charged residues exert their effect on topology. This work will be done with the E. coli leader peptidase, subunit H of the photosynthetic reaction center, procoat, lac permease, and outer membrane protein A. A variety of approaches will be used, including in vivo and in vitro techniques. Protease mapping and biochemical methods will determine how positive charges exert their effect on protein topology. These studies in protein membrane assembly will eventually help answer some of the fundamental questions in cellular biology concerning protein compartmentalization.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
9020759
Program Officer
Eve Ida Barak
Project Start
Project End
Budget Start
1991-04-01
Budget End
1994-09-30
Support Year
Fiscal Year
1990
Total Cost
$270,000
Indirect Cost
Name
Ohio State University
Department
Type
DUNS #
City
Columbus
State
OH
Country
United States
Zip Code
43210