Abstract
Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.
Highlights
Most inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP)1 pathway and insert into the membrane via the Sec machinery [1,2,3,4]
The major [35S] labeled product exhibits an Mr of about 33 kDa which is typical of LacY and is detected in the membrane fraction exclusively when ISO membrane vesicles are present during transcription/translation (Fig.1A)
In the absence of DDM, the band corresponding to full-length permease is completely digested, and a band corresponding to N6, as well as a band corresponding to C6 plus the biotin acceptor domain (BAD), are observed
Summary
Most inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP) pathway and insert into the membrane via the Sec machinery [1,2,3,4]. The Sec machinery is comprised of SecY, SecE and SecG proteins in the cytoplasmic membrane [6,7,8,9] and contributes to the topology of some membrane proteins [10]. It has been shown [11,12] that the Sec machinery and YidC exist as a complex, and several inner membrane proteins interact with YidC during insertion. Examples include folding of intermediates, interaction between helices during translation/insertion, the order and timing of helices exiting from the translocon into the lipid bilayer and topological determinants
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