Binding of H-2Kb-specific peptides to TAP and major histocompatibility complex class I in microsomes from wild-type, TAP1, and beta2-microglobulin mutant mice.
Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m 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(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m 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 beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m 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.beta2m heterodimers.
Wang
P
P
Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden.
Raynoschek
C
C
Svensson
K
K
Ljunggren
H G
HG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006183:H-2 Antigens / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008862:Microsomes, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000378:metabolism; D011485:Protein Binding; D001613:beta 2-Microglobulin / Q000235:genetics*
D002352:Carrier Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D015395:Histocompatibility Antigens Class I; D001613:beta 2-Microglobulin; D000255:Adenosine Triphosphate; D010047:Ovalbumin