A sugar chain at a specific position in the nascent polypeptide chain induces forward movement during translocation through the translocon

Abstract

Nascent polypeptide chains synthesized by membrane bound ribosomes are cotranslationally translocated through and integrated into the endoplasmic reticulum translocon. Hydrophobic segments and positive charges on the chain are critical to halt the ongoing translocation. A marginally hydrophobic segment, which cannot be inserted into the membrane by itself, can be a transmembrane segment depending on its downstream positive charges. In certain conditions, positive charges even 60 residues downstream cause the marginally hydrophobic segment to span the membrane by inducing the segment to slide back from the lumen. Here we systematically examined the effect of a core sugar chain on the fate of a marginally hydrophobic segment using a cell-free translation and translocation system. A sugar chain added within 12 residues upstream of the marginally hydrophobic segment prevents the sliding back and promotes forward movement of the polypeptide chain. The sugar chain apparently functions as a ratchet to keep the polypeptide chain in the lumen. We propose that the sugar chain is a third topology determinant of membrane proteins, in addition to a hydrophobic segment and positive charges of the nascent chain.

Membrane Protein Insertion at the Endoplasmic Reticulum

“…Thus, in this and related scenarios, TMDs can be inserted nonsequentially, with some being temporarily skipped and residing briefly in the ER lumen or cytosol. This has been demonstrated for artificially constructed polytopic TMDs, as which weakly recognized TMDs have been observed in both the ER lumen and cytosol (Goder et al 1999, Kauko et al 2010, Yamagishi et al 2011). Even in vivo, TMDs of native IMPs at least transiently access the cytosol and/or ER lumen during polytopic IMP biosynthesis (Cheng & Gilmore 2006).…”

“…Some accessory factors, such as signal peptidase and oligosaccharyl transferase, are required for the proper folding and maturation of IMPs and, in some cases, could also influence topology (Goder et al 1999, Yamagishi et al 2011). Others, such as TRAP, are stably associated with membrane-bound ribosomes (Ménétret et al 2000) and may directly influence the structure of the ribosome-translocon complex (Fons et al 2003, Ménétret et al 2000) and/or the functionality of the Sec61 translocon (Fons et al 2003).…”

“…These and other considerations mean that poorly recognizable TMDs that in isolation are not able to insert must nonetheless be inserted in the context of the native IMP (Enquist et al 2009, Lu et al 2000). One way to imagine how this occurs is if the local contributions that would normally disfavor insertion of a TMD can be overcome by strong constraints imposed by other parts of the protein (Fujita et al 2010, Kauko et al 2010, Nilsson et al 2000, Yamagishi et al 2011). …”

Adaptation of low-resolution methods for the study of yeast microsomal polytopic membrane proteins: a methodological review

“…A theoretical criticism of this approach derives from the fact that during translocation certain internal hydrophobic sequences can invert the in/out orientation of a preceding TM helix, and that the frequency of such an inversion is strongly reduced if an N-glycan is added to the loop lying between the two hydrophobic sequences [35,36]. Thus addition of N-glycoslation sites theoretically may induce abnormal topology, which may not be recognized as such if it occurs in parts of the proteins that are not functionally important.…”

A Few Positively Charged Residues Slow Movement of a Polypeptide Chain across the Endoplasmic Reticulum Membrane

“…In this process, simple partitioning of sufficiently hydrophobic segments into the lipid phase through the translocon is a major and rational motivation for the stop-translocation and membrane insertion of the transmembrane segments. , Positive charges on the nascent chain enhance the stop translocation. ,, The positive charges can be effective in vitro even when they are separated from the hydrophobic segment by more than 60 residues. In such cases, a marginally hydrophobic segment that had been translocated into the ER lumen can be retrieved to the membrane by the 60-residue downstream positively charge clusters, indicating that the positive charges not only fix the transmembrane segment at the membrane but also induce retrograde movement of the polypeptide chain. , Furthermore, we demonstrated that a cluster of 10 positively charged residues momentarily arrest the movement, even in the absence of a hydrophobic segment . Interestingly, the charge cluster can slowly move after the momentary stalling.…”