A novel complete reconstitution system for membrane integration of the simplest membrane protein

Biochem Biophys Res Commun. 2010 Apr 9;394(3):733-6. doi: 10.1016/j.bbrc.2010.03.061. Epub 2010 Mar 15.

Abstract

A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors. A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia coli. Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level. Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration. Based on the function of the factor, we propose that the factor is named MPIase (Membrane Protein Integrase).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Cysteine Endopeptidases / metabolism
  • Diglycerides / chemistry
  • Diglycerides / pharmacology
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / metabolism
  • Inovirus
  • Liposomes / chemistry
  • Liposomes / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism*
  • Protein Biosynthesis
  • SEC Translocation Channels

Substances

  • Capsid Proteins
  • Diglycerides
  • Escherichia coli Proteins
  • Liposomes
  • Membrane Proteins
  • SEC Translocation Channels
  • Cysteine Endopeptidases
  • mepS protein, E coli