J. Biol. Chem., Vol. 263, Issue 10, 4549-4560, Apr, 1988

Intracellular transport of class I histocompatibility molecules. Influence of protein folding on transport to the cell surface

DB Williams, F Borriello, RA Zeff and SG Nathenson
Department of Biochemistry, University of Toronto, Ontario, Canada.

To examine the structural requirements for the intracellular transport and surface expression of Class I histocompatibility molecules, we studied somatic cell variants that produce altered forms of the H-2Kb molecule. One variant, R8.10, produced a mutant Kb molecule that was expressed on the cell surface at about 15% of the wild-type level. Nucleic acid sequence analysis identified the mutation as a single nucleotide change resulting in an amino acid substitution (Trp----Arg) at residue 167 in the alpha 2 extracellular domain. A comparative kinetic analysis of the intracellular transport of the wild-type and mutant molecules revealed that transport of the mutant product was remarkably impaired. Whereas wild-type molecules arrived at the cell surface with a half-time of about 30 min, 80% of newly synthesized mutant molecules did not progress beyond the rough endoplasmic reticulum, where they were slowly degraded. Surprisingly, the remaining 20% of the mutant population was capable of reaching the plasma membrane at a rate about one-half that of wild-type Kb. The accumulation of most of the mutant molecules in the rough endoplasmic reticulum was not due to aggregation or insolubility, nor could it be attributed to a lack of association with beta 2-microglobulin. Several techniques were employed in an effort to detect structural features unique to the transport-deficient mutant population. Monoclonal antibody binding experiments revealed structural differences between wild-type and mutant molecules in the region of the mutation but failed to distinguish the transport-competent and -deficient mutant populations. Detergent partitioning studies were also ineffective in this regard. However, differences between transported and untransported molecules were readily demonstrated by their disparate susceptibilities to proteolytic digestion. The results indicated that the transport- deficient form of the mutant assumed a conformation that was altered relative to both the transport-competent form and the wild-type molecule. The fact that the ability of wild-type and mutant molecules to be transported correlated with conformational features rather than with their specific primary sequences suggests that proper folding is an important requirement for their passage through the exocytotic pathway.
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