Abstract
Recent studies have shown that the endoplasmic reticulum (ER)-resident protein, calnexin, associates with class I major histocompatibility complex (MHC) molecules early in their biosynthesis. It has been suggested that calnexin participates in the assembly of class I MHC molecules or in the retention within the ER of unassembled class I molecules. We have examined the role of phosphorylation of calnexin in its association with mouse class I MHC molecules. We show that phosphocalnexin associates with H-2Ld and H-2Db molecules but not with H-2Kb and H-2Dd molecules, although calnexin-H-2Kb association can be demonstrated. These observations are interesting in light of the fact that H-2Kb and H-2Dd molecules are transported out of the ER more rapidly than are H-2Ld and H-2Db molecules. H-2Ld and H-2Db molecules differ in amino acid sequence only in their membrane-distal alpha 1 and alpha 2 domains. Nevertheless, the affinity of phosphocalnexin for H-2Ld is greater than its affinity for H-2Db. Furthermore, H-2Db becomes endoglycosidase H-resistant more slowly in cells in which it associates with phosphocalnexin than in cells in which it does not. Ca2+ ionophore A23187 prevents association of phosphocalnexin with H-2Ld molecules in vivo but does not cause the disruption of phosphocalnexin-H-2Ld complexes after they have formed. A23187 does not prevent assembly of H-2Ld-beta 2-microglobulin (beta 2-m) heterodimers. Furthermore, phosphocalnexin is found associated with H-2Ld molecules regardless of their state of assembly with beta 2-m and antigenic peptide. These results suggest that phosphocalnexin association with class I MHC molecules does not play a role in assembly of the class I MHC-beta 2-m-peptide complex nor in preventing release of unassembled class I molecules from the ER but may otherwise influence their rate of transport through the ER.
Highlights
Recent studies have shown that the endoplasmic re- Pz-m (12 kDa) light chain, and an 8- or 9-amino acid peptide ticulum (ER)-resident proteinc,alnexin, associates with derivedfromself,viral,ortumorproteinsmadewithinthe class I major histocompatibility complex(MHC)mol- same cell [7,8,9]
I MHC heavy chains is prolongedceilnl lines lacking functional the affinity of phosphocalnexin for H-2Ldis greater than&-m or peptide transporter genes [14]. This findinghas been its affinity for H-2Db.H-2Dbbecomes en- interpreted to implythat calnexin interacts with incompletely doglycosidase H-resistant more slowly in cells in which assembled class I MHC molecules to retain them in the ER
Density gradient analysis suggests that H-2Ld molsultssuggest that phosphocalnexin association with ecules are bounbdpyhosphocalnexitnhroughoutht eir class IMHC molecules does not play a role in assembly of the class I MHC-P,m-peptide complex nor in preventing release of unassembled class I molecules from the ER but may otherwiseinfluence their rate of transport through the ER
Summary
(Received for publication, December 9, 1993, and in revised form, January 10, 1994). From the Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064. Recent studies have shown that the endoplasmic re- Pz-m (12 kDa) light chain, and an 8- or 9-amino acid peptide ticulum (ER)-resident proteinc,alnexin, associates with derivedfromself,viral,ortumorproteinsmadewithinthe class I major histocompatibility complex(MHC)mol- same cell [7,8,9] The assemblyof this trimolecular complex isa ecules early in theibriosynthesis. I MHC heavy chains is prolongedceilnl lines lacking functional the affinity of phosphocalnexin for H-2Ldis greater than&-m or peptide transporter genes [14] This findinghas been its affinity for H-2Db.H-2Dbbecomes en- interpreted to implythat calnexin interacts with incompletely doglycosidase H-resistant more slowly in cells in which assembled class I MHC molecules to retain them in the ER it associateswith phosphocalnexin thanin cells in until formation of the trimolecular complex is complete.
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