Different conformations of nascent polypeptides during translocation across the ER membrane

We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244).Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro).The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly α-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.Most eukaryotic secretory protein are translocated across the membrane of the endoplasmic reticulum (ER) through a continuous ribosome-translocon channel that is sealed to the cytoplasmic compartment and open towards the ER lumen [1]. Recent structural studies of ribosomes from Haloarcula marismortui suggest that the channel through the large ribosomal subunit is ~ 100 ？ long and 10-20 ？ wide [2], and the same appears to hold true also for yeast and rabbit ribosomes [3]. The internal diameter of the translocon channel present in dog pancreas microsomes has been estimated to be 40-60 ？ [4]. It is unclear to what extent proteins can fold already within the ribosome-translocon channel; studies on ribosome-nascent chain complexes suggest that only a limited degree of folding (s