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Vol. 20, Issue 2, 685-698, January 15, 2009

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Sequence-specific Retention and Regulated Integration of a Nascent Membrane Protein by the Endoplasmic Reticulum Sec61 Translocon

David Pitonzo^*, Zhongying Yang^*, Yoshihiro Matsumura^*, Arthur E. Johnson^,, and William R. Skach^*

*Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, Portland, OR 97239; Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114; and Departments of Chemistry, and Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843

Submitted September 3, 2008; Accepted November 12, 2008
Monitoring Editor: Peter Walter

A defining feature of eukaryotic polytopic protein biogenesis involves integration, folding, and packing of hydrophobic transmembrane (TM) segments into the apolar environment of the lipid bilayer. In the endoplasmic reticulum, this process is facilitated by the Sec61 translocon. Here, we use a photocross-linking approach to examine integration intermediates derived from the ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) and show that the timing of translocon-mediated integration can be regulated at specific stages of synthesis. During CFTR biogenesis, the eighth TM segment exits the ribosome and enters the translocon in proximity to Sec61. This interaction is initially weak, and TM8 spontaneously dissociates from the translocon when the nascent chain is released from the ribosome. Polypeptide extension by only a few residues, however, results in stable TM8-Sec61 photocross-links that persist after peptidyl-tRNA bond cleavage. Retention of these untethered polypeptides within the translocon requires ribosome binding and is mediated by an acidic residue, Asp924, near the center of the putative TM8 helix. Remarkably, at this stage of synthesis, nascent chain release from the translocon is also strongly inhibited by ATP depletion. These findings contrast with passive partitioning models and indicate that Sec61 can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.

Address correspondence to: William R. Skach (skachw{at}ohsu.edu).

Abbreviations used: aa, amino acid residue; ANB, N-(5-azido-2-nitrobenzoyl); RRL, rabbit reticulocyte lysate; TM, transmembrane.

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