Reconstitution of membrane proteolysis by FtsH.
Escherichia coli FtsH is a membrane-bound and ATP-dependent protease responsible for degradation of several membrane proteins. The FtsH action is processive and presumably involves dislocation of the substrate from the membrane to the cytosol. Although elucidation of its molecular mechanism requires an in vitro reaction system, in vitro activities of this enzyme against membrane protein substrates have only been assayed using detergent-solubilized components. Here we report on the construction of in vitro reaction systems for FtsH-catalyzed membrane protein degradation. A combination of two inverted membrane vesicles or of two proteoliposomes, one bearing the enzyme and the other bearing a substrate, was fused by polyethylene glycol 3350 treatment. Addition of ATP then resulted in degradation of the substrate. It was shown that FtsH can function in the process of membrane proteins degradation without aid from any other cellular factors.
Akiyama
Yoshinori
Y
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. yakiyama@virus.kyoto-u.ac.jp
Ito
Koreaki
K
D049069:ATP-Dependent Proteases; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011510:Proteolipids / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; D014357:Trypsin