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Abstract
Hydrophobic integral membrane proteins are synthesized in the aqueous environment of the cell cytoplasm and achieve a unique orientation in a specific cellular membrane. Many membrane proteins are initially synthesized as precursor molecules, with an additional 15 to 30 amino acid residues at the amino terminus that are enzymatically removed during assembly of the protein into the membrane. The complexity of intact cells has obscured the individual contributions of the nascent protein, the phospholipid bilayer, and other intracellular elements. This thesis describes both biochemical and genetic experiments designed to define the necessary components for insertion of a protein into a membrane.
M13 coat protein, the major protein of the M13 bacteriophage, is particularly well-suited to such experimental approaches. Prior to its assembly into virus, coat protein spans the E. coli inner membrane. It is initially made as a higher molecular weight precursor, procoat, and its insertion into the infected cell membrane is independent of its synthesis. In addition, enzymatic processing and assembly of coat protein into the membrane occur independently of any of the other nine viral gene products. Only host cell elements are used to catalyze this assembly event.
To define the necessary cellular factors, reconstitution of the assembly reaction from isolated components in vitro was demonstrated. Procoat was isolated from an in vitro cell-free translation reaction. Despite undergoing denaturation during isolation, procoat spontaneously re-folds to a water-soluble form that assembles into membranes and serves as substrate for purified E. coli leader peptidase. Vesicles prepared from purified lipids and leader peptidase convert purified procoat to coat that is properly inserted into the bilayer. These studies did not reveal any other soluble components that are required for procoat assembly into a membrane and suggested a critical role for the leader peptidase. To understand further the function of the leader peptidase in the cell, its gene was cloned into a plasmid vector. 100-fold overproduction was achieved and pure leader peptidase was obtained from transformed cells. In addition, the position of the leader peptidase gene on the E. coli chromosome was determined along with its nearest co-transducing selectable genes.
