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tbstract In this study, we have established that FtsY, the E. coil homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP-binding protein which displays intrinsic GTPase activity. GTP was found to influence the protease sensil ivity of FtsY indicative of a conformational change. FtsY mutated in the 4th GTP-binding consensus element displayed reduced GTP-binding and -hydrolysis which correlated with a reduced ability to interact with SRP. Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild-type FtsY. These observations sugzest that in E. coli GTP is important for proper functioning of ~tsY in protein-targeting.

The transport of bacterial outer membrane proteins to their destination might be either a one‐step process via the contact zones between the inner and outer membrane or a two‐step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D‐structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.The opportunistic pathogen Pseudomonas aeruginosa secretes many proteins into the extracellular medium. The secreted proteins are synthesized in the cytoplasm and have to pass both membranes of the cell envelope. Four main pathways for the secretion of proteins, usually referred to as the type I, II, III, and the autotransporter pathway, have been identified in P. aeruginosa (1-4). The majority of the exoproteins characterized is secreted via the type II pathway, also referred to as the general secretory pathway. In addition to elastase, which is the most abundant secreted protein, lipase, alkaline phosphatase, exotoxin A, two phospholipases C, the staphylolytic protease LasA, the chitin-binding protein CbpD, and a putative aminopeptidase are secreted via this pathway (2, 5-7).Proteins secreted via the type I or type III pathways are translocated across the two membranes of the cell envelope in a single step, without a periplasmic intermediate. In contrast, the type II pathway is a two-step mechanism. The first step is the translocation across the inner membrane, which is mediated by the Sec machinery. In the periplasm, which contains chaperones and folding catalysts, the exoproteins fold into a (near-)native conformation (8). For several proteins that are secreted via a type II mechanism (9 -11), including elastase of P. aeruginosa (12,13), it has been demonstrated that folding in the periplasm is essential for the subsequent translocation across the outer membrane to occur. In P. aeruginosa, the translocation of the periplasmic intermediates across the outer membrane is mediated by a machinery composed of at least 12 proteins, encoded by the xcp genes (for a review, see Ref. 2).To investigate the biogenesis of proteins secreted via the type II pathway of P. aeruginosa, we have chosen elastase as a model. This metalloprotease, which is encoded by the lasB gene (14), is produced as a preproprotein. The pre-part is the signal peptide, which directs the translo...