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

Abstract

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 Sec61alpha. 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-Sec61alpha 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 Sec61alpha can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.

Dissecting the physiological role of selective transmembrane-segment retention at the ER translocon

“…S4). Although crosslinking using BMH, or other such reagents, can never achieve complete efficiency, the synchronisation of stalled nascent chains and the analysis of only C-terminally tagged polypeptides ensures that this approach accurately reports the environment of the bulk of the P2X2 chains present in each reaction (see also Daniel et al, 2008;Ismail et al, 2006;Mothes et al, 1997;Pitonzo et al, 2009). …”

“…Only artificial termination of translation was able to trigger the release of the TM segment into the lipid phase and the delay of the Cterminal TM segments of opsin at the Sec61 complex is suggested to facilitate the assembly of two distinct folding domains within the protein . More recently, the eighth TM segment in the cystic fibrosis transmembrane conductance regulator (CFTR) protein was found to be specifically delayed at the ER translocon during CFTR biosynthesis, via a process that is dependent upon the presence of a charged residue within this membrane span (Pitonzo et al, 2009). The authors concluded that such retention might facilitate the formation of inter-TM segment contacts in CFTR necessary for the correct folding of the protein, and invoked the The membrane integration of polytopic proteins is coordinated at the endoplasmic reticulum (ER) by the conserved Sec61 translocon, which facilitates the lateral release of transmembrane (TM) segments into the lipid phase during polypeptide translocation.…”

“…active participation of the Sec61 complex in regulating this process (Pitonzo et al, 2009). Thus, although the phenomenon of TMsegment retention at the ER translocon is clearly an important facet of polytopic protein biosynthesis, the generality and importance of this process is presently poorly understood.…”

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

“…In this regard, two TM segments have been found to accumulate within or adjacent to the translocon before final integration into the lipid bilayer. [39][40][41][42][43] Furthermore, molecular dynamics simulations of the translocon in a fully solvated lipid bilayer showed that two helices can coexist within the translocon (J. Gumbart, personal communication). An interesting possibility arising from these observations is that the translocon may facilitate membrane protein integration by allowing efficient polar interactions between consecutive TM segments harboring charged residues and thereby stabilizing them in the nascent polypeptide.…”

Stitching proteins into membranes, not sew simple

“…Helical hairpin (consisting of two closely spaced helical TM segments separated by a short extra-membrane or surface turn) insertion is another possible mechanism for integrating marginally hydrophobic TM domains that relies on TM1 and TM2 achieving transmembrane location in a concerted manner rather than sequentially (Meacock et al, 2002;Sauri et al, 2005Sauri et al, , 2007Cross et al, 2009;Pitonzo et al, 2009). More recently, charge-pair interactions between a TM1 and a TM2 together with turn-promoting residues have been found to favour helical hairpin insertion (Bano-Polo et al, 2013), while neither the TM1 or the TM2 inserted efficiently independently of one another (Martinez-Gil et al, 2011a).…”