The enzymatically active form of protease 1, the major exocellular protein produced by Pseudomonas aeruginosa strain 34362, has been shown to exist exclusively exocellularly with no significant cell-associated activity. However, the presence of a cell-associated, enzymatically inactive protein which is serologically cross-reactive with, and convertible to, active enzyme has been demonstrated. One method of conversion of "precursor" to active enzyme is via limited proteolysis. Two assay systems for precursor were developed, one a radioimmune assay, and the other a proteolytic activation procedure. Localization studies suggest that the association while more tenacious than classical periplasmic enzymes is still an ionic rather than a covalent one. Kinetics of production studies showed to precursor to be synthesized early in the growth cycle and to accumulate prior to the rapid release of the active enzyme. Molecular weight studies showed only slight changes produced upon activation.

An inactive precursor to the active exocellular protease 1 of Pseudomonas aeruginosa is cell-associated and located primarily in the periplasmic space. We have studied factors that bring about activation of the precursor in vitro in order to shed some light on the process of its activation and secretion in vivo. A variety of diverse procedures were shown to effect irreversible activation. Several mild non-enzymatic procedures were effective, such as dialysis of an ammonium sulfate precipitate against neutral buffers, gel filtration (Sephadex G-loo), and ion-exchange chromatography (DEAE-cellulose). Activation also resulted following treatment with anionic detergents (sodium dodecyl sulfate, N-lauroyl sarcosine) and deoxycholate. Limited exposure to any of-several proteases with different specificities also resulted in activation. The kinetics of detergent-catalyzed activation reveals a long lag followed by rapid activation, suggesting at least a two-stage process. The precursor and the mature protease 1 have indistinguishable molecular masses (33 kDa), as measured by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis of these proteins purified by immunoabsorbance chromatography under denaturing conditions. Further, both precursor and protease have identical N-terminal alanine. Our results suggest that it is improbable that activation is the result of proteolytic processing of the precursor itself, but rather that it may involve the removal of a non-covalently associated inhibitor molecule. Hydrophobic interaction chromatography on octyl-Sepharose revealed that activation was accompanied by a significant change in the hydrophobicity, pointing to a significant change in the conformation of the precursor and the mature protease.A mutant has been studied which accumulates activatable precursor in the periplasm but releases no active enzyme into the culture medium, supporting the hypothesis that secretion through the inner and outer membranes proceed by different mechanisms. Comparison of outer membranes of protease-secreting strains (34 362A and PAKS 1) and a protease-negative mutant (PAKS 18) which accumulates precursor has shown that there is a change in the outer membrane protein profile in the latter.

A defined medium capable of supporting growth and exocellular protease production by clinical isolates of Pseudomonas aeruginosa has been developed. Control of protease production is effected by a mixture of three amino acids and glucose.

mRNA for prepro-a--factor (ppc), a yeast secretory glycoprotein, was translated in a wheat germ cell-free system that was posttranslationally supplemented either with inverted vesicles from the plasma membrane ofEscherichia coli (INV) or with microsomes from Saccharomyces cerevisiae. A postribosomal supernatant (PRS) from E. coli was found to stimulate translocation of ppa across the INV membrane. A yeast PRS could substitute for its E. coli counterpart. Likewise, an E. coli PRS could substitute for a yeast PRS and stimulate translocation of ppa across yeast microsomal membranes.The processes of secretory protein translocation across the prokaryotic plasma membrane and the eukaryotic endoplasmic reticulum (ER) have common features. Most strikingly, important structural and functional properties of the signal sequences of both prokaryotic and eukaryotic secretory proteins appear to have been conserved (1)(2)(3)(4)(5)(6)(7). However, it is not known whether or to what extent the corresponding signal recognition and translocation machineries resemble each other. Although genetic studies (for review see refs. 8 and 9) and biochemical studies (10, 11) indicate the existence of such machineries in prokaryotes, none of the components have so far been isolated. Thus, it is not known whether prokaryotes contain equivalents of the signal recognition particle (SRP) and its cognate receptor in the ER, the SRP receptor (for review, see ref. 12).Recently an extensively subfractionated cell-free system from Escherichia coli was developed in which translocation across inverted vesicles of the E. coli plasma membrane (INV) was demonstrated to be stimulated by a soluble factor or factors (10). Likewise, an activity present in the cytosol of yeast (Saccharomyces cerevisiae) has recently been shown to stimulate secretory protein translocation across yeast microsomal membranes (13). In this paper, we show that these prokaryotic and eukaryotic factors can substitute for each other in a cell-free translocation system containing INV or yeast microsomal membranes.METHODS AND MATERIALS SP6 Transcription. Plasmid pDJ100 containing prepro-afactor (ppa) structural gene (14) cloned in the BamHI site of the polylinker of pSP65 (15) was a gift of David Julius (Columbia University). Plasmid pLB8000 containing the structural gene for X phage receptor (16), LamB, cloned in the Sma I site of the polylinker of pSP64 was a gift of Spencer Benson (Princeton University). Plasma pDJ100 was linearized with Xba I, and plasmid pLB8000 was linearized with EcoRI prior to transcription. Transcriptions using the SP6 phage system were done as described in ref. 15.Wheat Germ Translation. Translations in the wheat germ system were done essentially as described by Erickson and Blobel (17). ppa mRNA (200 ng per 25 ,ul of translation mix) was translated in a staphylococcal nuclease-treated wheat germ system (8 ,ul of wheat germ extract per 25 ,ul of translation mix). The concentrations of KOAc and Mg(OAc)2 were adjusted to 112 mM and 2.1 mM, respectively ...