Protein secretion (including enzymes, immunoglobulins, protein hormones, and microbial toxins) is an essential function of cells. To try to identify components of the apparatus of protein secretion we have separated the membrane of B. subtilis into a complexed fraction (CM) carrying ribosomes and a free fraction, and these have been found to differ markedly in protein composition. Some of the unique CM proteins, identified and recovered by immunoprecipitation, are also found in the cytoplasm, complexed with several other proteins. We will test whether these complexes play a role in initiating secretion into membrane vesicles in vitro. In addition, we have subfractionated CM into preparations carrying single ribosomes or carrying polysomes, and differences in their protein composition suggest that we will be able to distinguish membrane proteins involved in initiation from another set involved in the later stages of protein secretion. We will use several tests to determine which of the unique CM proteins are involved in secretion: protection by attached ribosomes from digestion or labeling, cross-linking to ribosomes or to their nascent chains, and retention by ribosomes after extraction of membrane by non-denaturing detergent. The functions of these proteins will be studied in an in vitro secretion system with active membrane vesicles; in some cases with the aid of specific antibodies. Active membrane vesicles will be subfractionated, and their activities assessed. The ultimate aim is reconstitution of active membrane vesicles by incorporating the required membrane proteins into liposomes. The fractionation of the bacterial membrane is also useful for studying membrane differentiation and morphogenesis, and to this end we will also compare various membrane fractions with various functions, and examine the synthesis and localization of several membrane proteins such as penicillin-binding proteins.
NIH ExPorter
NIH ExPorter