Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli
Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.
- Research Organization:
- Nagoya Univ. (Japan)
- OSTI ID:
- 6464934
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 264:3
- Country of Publication:
- United States
- Language:
- English
Similar Records
Alleviation of CCCP-induced mitochondrial injury by augmenter of liver regeneration via the PINK1/Parkin pathway-dependent mitophagy
Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells. [Daucus carota Danvers]
Related Subjects
HYDRAZONES
BIOLOGICAL EFFECTS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
ATP
ATP-ASE
BIOASSAY
CELL MEMBRANES
CENTRIFUGATION
ESCHERICHIA COLI
INHIBITION
LIPOPROTEINS
PROTONS
ACID ANHYDRASES
BACTERIA
BARYONS
CELL CONSTITUENTS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HYDROLASES
LIPIDS
MEMBRANES
MICROORGANISMS
NUCLEONS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHOHYDROLASES
PROTEINS
SEPARATION PROCESSES
560300* - Chemicals Metabolism & Toxicology