Pivotal Role of Calnexin and Mannose Trimming in Regulating the Endoplasmic Reticulum-associated Degradation of Major Histocompatibility Complex Class I Heavy Chain

Cornelia M Wilson,Mark R Farmery,Neil J Bulleid

doi:10.1074/jbc.m000567200

Cornelia M Wilson, Mark R Farmery + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m000567200

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Abstract

We have established a mammalian semipermeabilized cell system that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. We show that degradation required unfolding of the protein and was cytosol- and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated, suggesting that an interaction with calnexin stabilized heavy chain. Stabilization of heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure degradation of heavy chain. When degradation or mannose trimming was inhibited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway.

Highlights

The ability of the endoplasmic reticulum (ER)1 to recognize and retain unassembled and malfolded proteins has been identified as an important regulatory mechanism to ensure that only correctly folded and assembled proteins are transported through the secretory pathway [1]
Immunoprecipitation of heavy chain with the monoclonal antibody HC10 [29] shows that the newly synthesized heavy chain is in excess (Fig. 1A, lane 5), the yield of complex thereby being limited by the availability of free ␤2 microglobulin
The results presented here go some way to resolving the first of these questions, extending previous studies [17, 18] that suggested a key role played by calnexin and mannose trimming in regulating the folding and degradation of glycoproteins

Summary

Introduction

The ability of the endoplasmic reticulum (ER) to recognize and retain unassembled and malfolded proteins has been identified as an important regulatory mechanism to ensure that only correctly folded and assembled proteins are transported through the secretory pathway [1]. One consequence of this “quality control” is that retained proteins are selectively degraded by a mechanism that is distinct from lysosomal degradation and has previously been suggested to take place within the ER [2] It is clear from a number of studies that this degradation takes place within the cytosol, is mediated by the proteasome, and requires dislocation of the polypeptide targeted for degradation from the ER to the cytosol Inhibition of the mannose trimming either by expression in ER mannosidase-deficient yeast strains [17] or by incubation of mammalian cells in the presence of deoxymannojirimycin [12, 18, 20, 21] results in stabilization of polypeptide chains that would normally be degraded. Our results highlight the pivotal role played by calnexin in regulating assembly and degradation

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