Protein Folding during Cotranslational Translocation in the Endoplasmic Reticulum

Abstract

To test how far into the protein-conducting channel of the translocon complex a nascent polypeptide domain must move before it can fold, we analyzed the folding of in vitro translated products of truncated mRNAs encoding the Semliki Forest virus capsid protease domain (Cp) during translocation into microsomes. Cp folded when the C-terminal linker connecting it to the peptidyltransferase center was 64 amino acids or longer. This means that to fold, Cp must exit the translocon channel. With an uncleaved signal sequence, about one out of four of the Cp domains could undergo folding with a C-terminal linker of only 38-66 amino acids. This suggested that the constraint imposed on folding by the translocon complex may be less stringent for signal-anchored membrane proteins.

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

“…Upon reaching the last available codon, the nascent chain therefore remains tethered to the ribosome via a covalent peptidyl-tRNA bond. These arrested RNCs provide a snapshot of cotranslational intermediates and allow one to study how proteins fold vectorially as they emerge from the ribosome (25,26,28,48). To generate ribosome-bound FP intermediates, we fused GFP (eCFP, eGFP, Venus, and Citrine) and RFP (mStrawberry, mCherry, DS Red, and mTangerine) variants to a C-terminal reporter protein (CFTR).…”

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

“…This suggests a largely extended conformation of the nascent polypeptide and a position of the OSTactive site close to the translocon (Whitley et al 1996;Kowarik et al 2002). OST complexes of yeast and mammalian cells were suggested to be associated with the translocation complex and to interact with the ribosome (Chavan et al 2005;Shibatani et al 2005;Harada et al 2009).…”

SS33410, an Inhibitor of V-ATPase, Blocks Intracellular Protein Transport of the VSV-G Protein in the Golgi Compartment

“…They are translocated across the endoplasmic reticulum (ER) membrane and transported along the membrane-enclosed organelles. [1][2][3][4] Transfer of protein from one compartment to the other is mediated by membrane vesicles. 5,6) Glycoproteins synthesized on rough endoplasmic reticulum (rER) are intracellularly transported to their ultimate sites of residence via the Golgi apparatus.…”