Development of a functional in vitro integration system for an integral membrane protein, SecG

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Abstract

A functional in vitro integration system for an integral membrane protein, SecG, comprising an efficient translation system supplemented with inverted membrane vesicles (IMV) was developed. When SecG was synthesized in the presence of IMV prepared from a ΔsecG strain (ΔSecG IMV), the synthesized SecG was recovered with the IMV. A population of SecG was resistant to urea extraction, indicating that the synthesized SecG was integrated into ΔSecG IMV. Addition of signal recognition particle and its receptor (SRP) and SecA caused an increase in the amount of the urea-resistant form of SecG. When IMV into which SecG had been integrated were subjected to the translocation assay, the translocation activity was found to be significantly stimulated compared with for ΔSecG IMV. Moreover, when SRP and SecA had been supplemented, the translocation activity nearly recovered to the level in IMV prepared from the wild type strain. These results indicate that the in vitro synthesized SecG could be functionally integrated into ΔSecG IMV with the help of SRP and SecA. We also present evidence that the membrane targeting and integration of SecG is stimulated by externally added SecA and SecG itself.

Introduction

Most integral membrane proteins are thought to be targeted to and integrated into membranes cotranslationally. Signal recognition particle (SRP) and its receptor, and the protein conducting channel are responsible for membrane targeting and integration, respectively (for reviews, see [1], [2]). In Escherichia coli, SRP, composed of Ffh and 4.5 S RNA (a gene product of ffs), and FtsY, the receptor, are localized both in the cytosol and on the inner membrane. The SecYEG complex forms the protein conducting channel in the inner membrane. In addition, SecA, a translocation ATPase, is necessary for integration if membrane proteins possess a hydrophilic, periplasmic region to be translocated [3]. In vitro systems for membrane protein integration have been extensively developed to clarify the molecular mechanisms underlying membrane integration, however, the functional expression of membrane proteins has not been sufficiently examined. Moreover, it is rather difficult to analyze their functions after in vitro synthesis and integration, since for the conventional integration assays, membrane proteins are usually synthesized in radiochemical amounts.

Recent development of in vitro translation systems allows functional and structural analyses of proteins because of the sufficient synthesis levels. However, it remains difficult to obtain membrane proteins in a functional form with such systems due to their highly hydrophobic nature. Although the problem was overcome by detergent and/or lipid addition in some studies [4], [5], [6], [7], [8], numerous examinations of conditions are necessary for each protein. Alternatively, the functional integration of membrane proteins into membrane vesicles, which reflects the in vivo reaction correctly, is desirable as a versatile method, especially in the case of membrane proteins with unknown functions. Therefore, we combined an efficient translation system with the membrane integration reaction. We chose SecG as the substrate because we have extensively studied the SecG function, which can be easily monitored. SecG is a subunit of SecYEG and possesses two transmembrane stretches with both N- and C-termini exposed to the periplasm ([9], see Fig. 3B). SecG stimulates protein translocation significantly under certain conditions [10], [11]. We report here that inner membrane vesicles (IMV) prepared from the ΔsecG strain were transformed into the wild type (wt) IMV through integration of in vitro synthesized SecG. We also report that SecA and SecG itself are involved in both the membrane targeting and integration of SecG.

Section snippets

Materials and methods

Bacterial strains and plasmids. K003 [12] and its ΔsecG derivative, KN553 [9], were used for IMV preparation. Plasmid pOAD26 was used to synthesize proOmpA D26, a truncated derivative of proOmpA [13]. Plasmid pIVEX-SecG, in which the secG gene is encoded under the control of the T7 promoter, was constructed by cloning an NdeI–SmaI fragment amplified using a primer pair (AAACATATGTATGAAGCTCTTTTAGTAG/ATTCCCGGGTTAGTTCGGGATATCGCTGG; the restriction sites are italicized, and the initiation and

SecG in vitro synthesized with the RTS system is integrated into ΔSecG IMV

SecG was synthesized by means of the RTS system in the presence of ΔSecG IMV. After the translation reaction, the IMV, recovered by sedimentation, were subjected to immunoblot analysis to check the SecG synthesis (Fig. 1A). An immuno-decorated band was clearly observed after the reaction (lanes 2 and 3), which could not be detected before synthesis (lane 4). The amount of the synthesized SecG was several-fold higher than that of wt IMV (lane 1). When SRP (Ffh and 4.5 S RNA as SRP, plus its

Discussion

In this study, we demonstrated that SecG could be in vitro integrated functionally, and that SRP/SecA addition stimulated both the targeting and integration steps, leading to functional expression. Our system would be applicable to the in vitro synthesis of functional membrane proteins in general. It has also been reported that in vitro synthesized SecY was integrated into IMV in a functional form [24], however, the activity recovery was not sufficient due presumably to the low synthesis level.

Acknowledgments

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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