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Volume 271, Number 40, Issue of October 4, 1996 pp. 24830-24835
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Binding of H-2K^b-specific Peptides to TAP and Major Histocompatibility Complex Class I in Microsomes from Wild-type, TAP1, and ₂-Microglobulin Mutant Mice

(Received for publication, April 15, 1996, and in revised form, June 18, 1996)

Ping Wang , Carina Raynoschek , Kerstin Svensson and Hans-Gustaf Ljunggren ^¶

From the  Ludwig Institute for Cancer Research, Stockholm Branch, Box 240 and the ^¶ Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden

Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), ₂-microglobulin (2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either 2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1^/, 2m^/, and TAP1/2m^/ mice using a cross-linkable H-2K^b-binding peptide. This enabled us to study the influence of an intact TAP complex and 2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1^/ mice. In contrast, no peptide binding to 2m-free HC was detected in permeabilized microsomes from 2m^/ and TAP1/2m^/ mice. However, the addition of exogenous 2m allowed peptide binding to class I in permeabilized 2m^/ and TAP1/2m^/ microsomes. These results demonstrate that a preformed class I HC·2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1^/ microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC·2m heterodimers.

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