Blocking the later stages of the pathway for the export of outer membrane proteins by E. coli leads to an inhibition of expression of these proteins which appears to be at the level of translation. This appears to be coupled to the later stages of export. These later stages will be studied in three ways. First, using oligonucleotide-directed mutagenesis mutations will be introduced into a C-terminal region of the OmpC porin protein which has been identified as a site of trans-dominant mutations which blocks export. The effects of these mutations on secretion, protein folding and oligomer stability will be examined. Attempts will be made to set up an in vitro system for folding and trimerization of OmpC protein using protein and LPS precursors which approximate thos found in vivo. This should allow additional study of mutant proteins, and can be used to study protein-LPS interaction during assembly. Third, studies on the mechanism of export-coupled regulation by using signal sequence mutations and protein fusions in both OmpC and LamB to identify protein structural requirements to be either an initiator or a target of this regulatory control. There is a growing appreciation in the field of protein export that the dog pancreas microsome-wheat germ extract system which yielded the discovery of SRP and docking protein and so much other information is in fact a very atypical system since it is primarily co-translational. Export into mitochondria and out of both bacteria and yeast seems to be primarily post- translational, and evidence for a particle analogous to SRP is lacking. Thus the SRP-mediated elongation arrest can not operate in these systems, and if there is regulation coupled to export it must operate in a different fashion. This group seems to have a handle on an alternative regulatory system which, although specialized, may be a prototype for many other systems. This project is an attempt to define some of the basic parameters of this regulatory system as well as to examine the later steps in E. coli outer membrane protein export pathway such folding and interaction with small ligands.
