The Role of ERp57 in Disulfide Bond Formation during the Assembly of Major Histocompatibility Complex Class I in a Synchronized Semipermeabilized Cell Translation System

Mark R Farmery,Simon Allen,Amanda J Allen,Neil J Bulleid

doi:10.1074/jbc.275.20.14933

Abstract

We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly.

Highlights

Complex and inter-related quality control mechanisms exist within the lumen of the mammalian endoplasmic reticulum (ER)1 to ensure correct protein folding
Once a heavy chain-␤2-microglobulin dimer is formed, this dissociates from calnexin and associates with calreticulin, transporter associated with antigen presentation (TAP), and tapasin to form a complex that is required for peptide loading [3, 6, 7]
ERp57 has been shown to interact with major histocompatibility complex (MHC) class I heavy chain, ␤2-microglobulin, calreticulin, and tapasin in a subcomplex that is dependent on the presence of ␤2-microglobulin and tapasin but not TAP [30]

Summary

MHC Class I Assembly

The interaction of ERp57 with MHC class I in comparison with calnexin. In human cells, ERp57 has been shown to interact with MHC class I heavy chain, ␤2-microglobulin, calreticulin, and tapasin in a subcomplex that is dependent on the presence of ␤2-microglobulin and tapasin but not TAP [30]. To study what is clearly a complex process, one would ideally investigate folding and assembly within a functionally intact ER To address this aim we have developed techniques that will allow us to delineate the time course of folding, assembly, and interaction of MHC class I molecules with ER resident proteins. This approach makes use of semipermeabilized cells (SP cells) that can be prepared from any cell line grown in culture and added to an in vitro translation system where they act as a source of ER [14]. We show that the recently discovered glycoprotein-specific chaperone ERp57 [15] interacts with heavy chain during disulfide bond formation

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Methods