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

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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).

Introduction

Integral membrane proteins are integrated into biological membranes in a cotranslational manner with functions of integration factors. In Escherichia coli, several pathways are utilized for integration (for review, see [1], [2]). Nascent chains carrying hydrophobic membrane spanning stretches are recognized by a signal recognition particle (SRP) composed of Ffh and 4.5 S RNA, followed by targeting to membrane by means of an SRP receptor, FtsY. Integration into membrane occurs at SecYEG, a protein conducting channel. On the other hands, several small membrane proteins are unable to utilize SRP system. Thus, these proteins are independent of both the SRP system and SecYEG. Moreover, these proteins are integrated into liposomes composed of phospholipids. Based on these observations, they have long been thought to integrate spontaneously. However, we found that diacylglycerol (DAG), which is contained in native membranes, completely blocks such spontaneous integrations at a concentration of physiological level, and that integration strictly depends on an integration-stimulating factor [3], [4]. This factor is involved not only in integration of SRP/Sec-dependent proteins but also in translocation of presecretory proteins [3]. Hereafter, we call the factor as MPIase (Membrane Protein Integrase), based on its function.

The PURE system is an in vitro translation system composed of the defined purified factors involved in translation in E. coli[5]. The usage of PURE system enables us not only to obtain large amounts of translation products reproducibly, but also to critically examine the involvement of cytosolic factors. Therefore, development of the in vitro synthesis of membrane proteins and integration into liposomes only containing the necessary factors for integration is preferable for the functional and structural analysis of membrane proteins. In these analyses the integration system that reflects in vivo reactions would be better, especially when membrane proteins with unknown functions are examined. In this paper, we developed further purification scheme to obtain the highly purified MPIase, followed by the establishment of the complete reconstitution system for integration of 3L-Pf3 coat by the combination of PURE system with MPIase-containing liposomes.

Section snippets

Materials

Inverted inner membrane vesicles (INVs) were prepared from MC4100 (F Δ[argF-lac]U169 araD rpsL150 relA1 thi deoC7 ptsF25 flbB5301[6]), as described [7]. Plasmid T7-7-3L-Pf3 encoding 3L-Pf3 coat [8] was kindly provided by Prof. Andreas Kuhn. E. coli polar phospholipids and 1,2-dioleoyl-sn-glycerol were purchased from Avanti polarlipids. Tran35S label, a mixture of [35S]methionine and [35S]cycteine (>37.0 TBq/mmol), was from MP Biochemicals. Proteinase K and TLC plates were from Merck.

Purification of MPIase

MPIase was further purified by partition chromatography because TLC analysis revealed that previously purified preparation was contaminated by lipopolysaccharide (LPS). On the TLC plate visualized by the anisaldehyde reagent, the active preparation after the MonoQ step gave two spots, brown one with mobility of ∼0.35 of Rf value and gray one with slightly lower mobility (Fig. 1A, lane 1). Since Ra-LPS prepared from MC4100 gave only a gray spot of the same mobility as the gray one (data not

Discussion

In this paper, we demonstrated that the complete reconstitution of integration of the simplest membrane protein was successfully developed, which reflects the in vivo reaction correctly. In the system, we found that no cytosolic factors other than factors involved in translation are necessary and that as a membrane component only MPIase is necessary under the conditions where no spontaneous integrations occur. We have chosen 3L-Pf3 coat as a substrate protein, which is the simplest membrane

Acknowledgments

We thank Prof. Andreas Kuhn for the gift of the plasmid. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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