The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ⌬H ϩ . In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

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Studies were performed to unravel the activation and maturation mechanism of vacuolar (lysosomal) proteinases in Saccharomyces cerevisiae. In vivo and in vitro studies show that proteinase yscA and proteinase yscB are involved in the activation and processing event of pro‐carboxypeptidase yscY. Processing and activation of pro‐carboxypeptidase yscY by proteinase yscA depends on an additional factor contained in the vacuolar fraction. Comparable activation can be mimicked by sodium polyphosphate. Optimum pH for processing by this proteinase yscA‐triggered event is 5. The proteinase yscA‐triggered maturation process of pro‐carboxypeptidase yscY leads to an intermediate mol. wt form of the enzyme which is, however, fully active. Proteinase yscB transfers the intermediate mol. wt form of the original precursor to the apparently authentic, mature and active carboxypeptidase yscY. An activation and maturation scheme is devised.

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Yeast mutants lacking proteinase A were isolated. One of these mutants (Hb I) is characterized in detail. The mutation called pral segregates 2: 2 in meiotic tetrads indicating a single gene mutation. N o anti-(proteinase A) cross-reacting material can be detected. Diploids heterozygous for praI show gene dosage. Thus, it appears that PRAl might be the structural gene for proteinase A.Results obtained with this mutant show that proteinase A is not a vital component of the vegetative cell cycle. The mutant exhibits normal mitotic growth under rich and poor growth conditions and shows normal mating. Enzymes subject to carbon catabolite inactivation and inactivation of NADP-dependent glutamate dehydrogenase, processes which were proposed to be of proteolytic nature, are not affected by the absence of proteinase A. However, protein degradation under sporulation conditions is about 30 ; ( reduced in proteinasc A mutant cells. The differentiation process of spordation is also disturbed leading to a 40 reduced sporulation frequency in mutant cells.