DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.

Abstract

In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+‐ATPase inhibitor N,N′‐dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co‐ and post‐translational translocations, suggesting an involvement of the H+‐translocating F1F0‐ATPase in either mode of transport. This was verified by (i) the dependence of efficient co‐translational translocation upon a low salt, i.e. F1‐containing extract from membrane vesicles; (ii) the co‐purification of the translocation activity present in this extract and F1‐ATPase; (iii) the inability of either vesicles or their low‐salt extract, derived from F1F0‐ATPase‐lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP‐dependent, post‐translational translocation into F1‐deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+‐ATPase and the other an as yet unidentified translocation factor.

Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli

“…Removal of the F 1 -ATPase from INV by low salt stripping impairs translocation of Sec precursors by the dissipation of the H ϩ -motive force and is reversed by the readdition of F 1 -ATPase or the low salt extract (LSE) (28). Likewise (Fig.…”

Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY

“…Using a cell-free system, we previously demonstrated translocation across and integration into the lipid bilayer of INV from E. coli of a variety of periplasmic, outer-membrane (Miiller and Blobel, 1984;Miiller et al, 1987;Krishnabhakdi and Miiller, 1988) and inner-membrane proteins (Ahrem et al, 1989). As recently reported, translocation by membrane vesicles is inhibited by a previous exposure to trypsin due to loss of precursor targeting (Swidersky et al, 1990).…”

“…Published procedures were employed for cell-free transcription/translation of plasmid DNA using an E. coli cell extract (Miiller et al, 1987;Swidersky et al, 1990; both variations were tested with each extract and plasmid for optimal synthesis activity). Preparation of INV, trypsin treatment of INV, subfractionation of translation products on a two-step sucrose gradient, translocation of the bound preLamB into membrane vesicles, SDS/PAGE (SecY-containing samples were denatured at 37°C) and fluorography, and quantitative determination of radioactivity contained in bands on SDS/ polyacrylamide gels were performed as previously described (Swidersky et al, 1990).…”

“…The plasmids used were pBR322 (bla), pLB7012 (IumB; Miiller et al, 1987), pN01573 (bla and secY; Akiyama and Ito, 1985), pLB8000 (IamB) and pLB8012 (lamBI2D). To construct pLB8000, plasmid HC2 carrying the lamB gene and flanking sequences (Clement and Hofnung, 1981) was digested with restriction enzymes SalI/XmaI and XmaIIStuI, and the DNA fragments were separated by agarose-gel electrophoresis.…”

“…It is essential for the export of periplasmic and outer-membrane proteins of Escherichia coli since mutations in the sec Y gene result in temperature-sensitive export defects, as shown both in vivo (Ito et al, 1983Shiba et al, 1984) and in vitro (Fandl and Tai, 1987;Baba et al, 1990). Homologues ofsecY have also been found in Bacillus subtilis (Suh et al, 1990), Mycoplasma capricolwn (Ohkubo et al, 1987) and Lactococcus lactis (Koivula et al, 1991). Distinct mutations in E. coli secY (designated prlA) suppress the export defects of various signal-sequence mutations (Emr et al, 1981 ;Emr and Bassford, 1982;Stader et al, 1986;Sako and Iino, 1988;Puziss et al, 1992), suggesting a, presumably transient, recognition by SecY of exported proteins via their signal sequences.…”