Investigating cotranslational protein integration into the endoplasmic reticulum membrane
McCormick, Peter Joseph
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During co-translational integration, the transmembrane (TM) sequence of a nascent membrane protein moves laterally into the ER lipid bilayer upon reaching the translocon. Our lab has previously shown that this movement is a multistep process, but it was not clear whether the observed photocrosslinking of the TM segment to translocon proteins resulted from specific interactions or simply from TM-translocon proximity. If the latter, the TM α-helix will be oriented randomly with respect to translocon proteins, whereas, if the former, a specific TM helix surface would face TRAM and/or Sec61α. Integration intermediates were prepared by in vitro translation of truncated mRNAs in the presence of a Lys-tRNA analog with a photoreactive moiety attached to the lysine side-chain. When photoadduct formation was monitored as a function of probe location within the TM α-helix, we found that the extent of photocrosslinking to TRAM and Sec61α was non-random. Thus, the TM sequence occupies a distinct location within the translocon, a result that can only be achieved through protein-protein interactions that mediate the lateral movement, positioning, and integration of the TM sequence. In the case of multi-spanning membrane proteins, it was unknown how multiple hydrophobic regions integrated into the ER membrane. By placing photoprobes within each of several TM domains of a multi-spanning membrane protein, we were able to determine at what stage of integration each TM segment was no longer adjacent to translocon proteins. Using this approach we were able to establish a mechanism of integration for multi-spanning membrane proteins co-translationally inserted into the ER membrane.