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Claudio A. Mosse^*, Leslie Meadows, Chance J. Luckey^*, David J. Kittlesen, Eric L. Huczko^*, Craig L. Slingluff, Jr., Jeffrey Shabanowitz, Donald F. Hunt^,||, and Victor H. Engelhard^*

From the ^* Department of Microbiology and the Beirne Carter Center for Immunology Research; the Department of Chemistry; the Department of Surgery; and ^|| the Department of Pathology, University of Virginia, Charlottesville, Virginia 22904

Formation of major histocompatibility complex class I–associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201–associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

¹ Abbreviations used in this paper: BFA, brefeldin A; ER, endoplasmic reticulum; m/z, mass/charge ratio; TAP, the transporter associated with antigen.

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