Detergent disruption of bacterial inner membranes and recovery of protein translocation activity

Journal Article · Wed Nov 01 04:00:00 EST 1989 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.86.22.8673· OSTI ID:7165632

Cunningham, K; Wickner, W T ^[1]

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1. Univ. of California, Los Angeles (USA)

Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. The authors treated inner-membrane vesicles of Escherichia coli with mixtures of octyl {beta}-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure ({sup 35}S)pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components.

• OSTI ID: 7165632
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 86:22; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADDITIVES
ATP
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DETERGENTS
ELECTROPHORESIS
EMULSIFIERS
ENZYMATIC HYDROLYSIS
ENZYMES
ESCHERICHIA COLI
ESTERS
EVEN-ODD NUCLEI
HYDROLASES
HYDROLYSIS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPIDS
LYSIS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHOLIPIDS
PROTEINS
RADIOISOTOPES
SOLVOLYSIS
SULFUR 35
SULFUR ISOTOPES
SURFACTANTS
WETTING AGENTS