Abstract
Membrane protein translocation and insertion is a central issue in biology. Here we focus on a minimal system, the membrane insertase YidC of Escherichia coli that inserts small proteins into the cytoplasmic membrane. In a reconstituted system individual insertion processes were followed by single-pair fluorescence resonance energy transfer (FRET), with a pair of fluorophores on YidC and the substrate Pf3 coat protein. After addition of N-terminally labeled Pf3 coat protein a close contact to YidC at its cytoplasmic label was observed. This allowed to monitor the translocation of the N-terminal domain of Pf3 coat protein across the membrane in real time. Translocation occurred within milliseconds as the label on the N-terminal domain rapidly approached the fluorophore on the periplasmic domain of YidC at the trans side of the membrane. After the close contact, the two fluorophores separated, reflecting the release of the translocated Pf3 coat protein from YidC into the membrane bilayer. When the Pf3 coat protein was labeled C-terminally, no translocation of the label was observed although efficient binding to the cytoplasmic positions of YidC occurred.
Highlights
A fundamental question of membrane protein insertion is how the hydrophilic parts of a protein are translocated across the membrane and how its hydrophobic parts are properly oriented and inserted into the lipid bilayer [1,2]
This study shows that membrane translocation of the Nterminal domain of the Pf3 coat protein occurs within milliseconds resulting in a close transient contact with all 3 periplasmic loops of YidC
We have shown that the orientation of the Pf3 coat protein is strictly determined by the charged residues present in the two hydrophilic regions; in the wild type the N-terminus is always in the periplasm [19]
Summary
A fundamental question of membrane protein insertion is how the hydrophilic parts of a protein are translocated across the membrane and how its hydrophobic parts are properly oriented and inserted into the lipid bilayer [1,2]. Membrane insertion of small bacterial, chloroplastic and mitochondrial proteins is catalyzed by members of the Oxa-1 family, like YidC of E. coli [2,3]. YidC is an essential protein and mutations in the YidC homologues of animals and plants cause severe defects in mitochondria [4] and chloroplasts [5]. YidC functions either alone in the so-called YidC-only pathway or in conjunction with the Sec translocase [3]. In contrast to the Sec translocase, YidC consists of a single protein component making it an ideal minimal system for in vitro studies. The periplasmic loops P1, P2 and P3 consist of 354, 23 and 3 amino acid residues, respectively
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