INVIVO ANALYSIS OF INTEGRATION OF MEMBRANE-PROTEINS IN ESCHERICHIA-COLI

The in vivo process of membrane protein integration was studied by pulse-labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction. To conduct this experiment, conditions for extracting E. coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins. Examination of pulse-labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome o subunit II and leader peptidase revealed that all were in the alkali-insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild-type cells. However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali-soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration. The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient. The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells. LacY overproduction in wild-type cells was found to inhibit processing (export) of beta-lactamase but not of OmpA and OmpF. These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of beta-lactamase.