Structure and folding of nascent polypeptide chains during protein translocation in the endoplasmic reticulum.

Robin L Haynes,Christopher V Nicchitta,Tianli Zheng

doi:10.1074/jbc.272.27.17126

Robin L Haynes, Christopher V Nicchitta + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.27.17126

Copy DOI

Abstract

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo and in vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.

Highlights

Current models of protein translocation across the mammalian endoplasmic reticulum (ER)1 depict translocation as a process in which vectorial transport accompanies formation of a tight junctional complex between the ribosome and the protein conducting channel with the free energy for translocation provided by passive diffusion [1, 2]
The analysis of the energetics of protein translocation is made difficult by the fact that protein translation, translocation, and folding are coincident processes and that protein folding occurs in an environment, the ER lumen, which is highly enriched in molecular chaperones and protein folding enzymes
On the basis of these data, we propose that lumenal protein-nascent chain interactions are paramount to efficient translocation and in their absence, irreversible protein-protein aggregation may serve as the predominant protein folding end point

Summary

Introduction

Current models of protein translocation across the mammalian endoplasmic reticulum (ER) depict translocation as a process in which vectorial transport accompanies formation of a tight junctional complex between the ribosome and the protein conducting channel with the free energy for translocation provided by passive diffusion [1, 2]. Dition of N-linked oligosaccharides, that occur coincident with translocation [7,8,9,10,11] In the latter model, interactions between the nascent chain and lumenal molecular chaperones are thought to prevent retrograde transport through the translocation pore and bias movement of the nascent chain into the lumenal compartment [3,4,5,6, 12]. The identification of the Sec61p complex as the primary ribosome receptor and translocation channel suggests that ribosome association with Sec61p could provide the aqueous pathway for nascent chain transit into the lumen [15, 16] It is clear from molecular genetic and biochemical studies that hsp proteins perform an essential function in protein translocation across the yeast ER and the mitochondrial inner membrane [17,18,19,20,21,22]. To study the contribution of protein folding and lumenal chaperone-nascent chain interactions to translo-

Methods

Results

Conclusion