Peptide-induced stabilization and intracellular localization of empty HLA class I complexes.
The human cell line T2 has been reported to be class I assembly deficient, and accordingly expresses reduced amounts of HLA-A2 and no HLA-B5 at the cell surface. By immunoblotting we observe the steady-state class I heavy chain levels of T2 to be near normal when compared with the identical class I alleles of the wild-type cell line T1. In pulse chase experiments, formation of heavy chain beta 2-microglobulin complexes is observed for both HLA-A2 and HLA-B5. Culture at reduced temperatures (26 or 20 degrees C) does not increase the amount of class I molecules transported, unlike what has been reported for the class I assembly-deficient mouse mutant cell line RMA-S. The HLA-B5 and the HLA-A2 complexes formed by T2 are thermolabile in cell lysates, albeit to different degrees. The thermolability of HLA-B5 can be overcome by addition of HLA-B5-presentable peptides, obtained by trifluoroacetic acid extraction from an HLA-B5-positive cell line, underlining the necessity of peptide for class I stability and indicating that T2-derived class I complexes are devoid of peptide. Cytoplast fusion of T2 cells with RMA-S cells shows the defect in class I assembly of RMA-S to be similar to that of T2. Localization of class I molecules observed by immuno-electron microscopy reveals the accumulation in the T2 cell line of both HLA-B5 and HLA-A2 in the endoplasmic reticulum (ER). Class I molecules are present in all the cisternae of the Golgi complex of T2, but the ratio of HLA-A and -B locus products in the Golgi area differs significantly from that at the cell surface. We conclude that the requirement for peptide in transport of class I molecules manifests itself at a stage beyond the ER, most likely the Golgi area.
Baas
E J
EJ
Department of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
van Santen
H M
HM
Kleijmeer
M J
MJ
Geuze
H J
HJ
Peters
P J
PJ
Ploegh
H L
HL
D002460:Cell Line; D015789:HLA-A2 Antigen / Q000032:analysis; D015235:HLA-B Antigens / Q000032:analysis; D015395:Histocompatibility Antigens Class I / Q000032:analysis*; D006801:Humans; D016253:Microscopy, Immunoelectron; D013696:Temperature; D001613:beta 2-Microglobulin / Q000032:analysis
D015789:HLA-A2 Antigen; D015235:HLA-B Antigens; C035270:HLA-B5 antigen; D015395:Histocompatibility Antigens Class I; D001613:beta 2-Microglobulin