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 colt 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 MPlase (Membrane Protein Integrase). (C) 2010 Elsevier Inc All rights reserved