MISFOLDED MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I MOLECULES ACCUMULATE IN AN EXPANDED ER-GOLGI INTERMEDIATE COMPARTMENT

Misfolded membrane proteins are rapidly degraded, often shortly after their synthesis and insertion in the endoplasmic reticulum (ER), but t he exact location and mechanisms of breakdown remain unclear. We have exploited the requirement of MHC class I molecules for peptide to achi eve their correct conformation: peptide can be withheld by introducing a null mutation for the MHC-encoded peptide transporter: TAP. By with holding TAP-dependent peptides, the vast majority of newly synthesized class I molecules fails to leave the endoplasmic reticulum and is deg raded. We used mice transgenic for HLA-B27 on a TAP1-deficient backgro und to allow visualization by immunoelectron microscopy of misfolded H LA-B27 molecules in thymic epithelial cells. In such HLA trans genic a nimals, the TAP mutation can be considered a genetic switch that allow s control over the extent of folding of the protein of interest, HLA-B 27, while the rate of synthesis of the constituent subunits remains un altered, In TAP1-deficient, HLA-B27 transgenic animals, HLA-B27 molecu les fail to assemble correctly, and do not undergo carbohydrate modifi cations associated with the Golgi apparatus, such as conversion to End oglycosidase H resistance, and acquisition of sialic acids. We show th at such molecules accumulate in an expanded network of tubular and fen estrated membranes. This compartment has its counterpart in normal thy mic epithelial cells, and is identified as an ER-Golgi intermediate, W e detect the presence of ubiquitin and ubiquitin-conjugating enzymes i n association with this compartment, suggesting a nonlysosomal mode of degradation of its contents.