Blocked transport of soluble K(b) molecules containing connecting peptide segment involved in calnexin association

Abstract

The molecular event governing the assembly of the MHC class I heavy chain-β2-microglobulin-peptide complex is still not fully understood. In order to characterize the transport properties of MHC class I molecules, several truncated H-2Kb genes were constructed and expressed in COS7 cells. Surprisingly, the expressed soluble molecule containing connecting peptide (CP) segment (sKb(CP)) did not secrete as efficiently as the one without CP (sKb(CYT)). When the sKb(CP) gene was transfected into a calnexin-deficient cell line CEM.NK(R), the amount of soluble Kb molecules in the supernatant was comparable with sKb(CYT)-transfected CEM.NK(R). To further demonstrate the different transport of sKb(CP) and sKb(CYT) within living cells, we attached green fluorescent protein (GFP) to the C-termini of both molecules and, as a comparison, to the full-length transmembrane counterpart (mKb-GFP). While the mKb-GFP-transfected cells showed the green fluorescence in the reticular network and the nuclear envelope, sKb(CP)-GFP showed obviously lump fluorescence of high intensity within cells. However, the distribution of sKb(CYT)-GFP was fairly uniform. Furthermore, GFP-tagged molecules allow us to analyze their interaction with other proteins in a direct, simple and quantitative method, designated immunofluorescence precipitation. The results showed that 60% of sKb(CP)-GFP molecules were associated with calnexin, while <10% with tapasin. Taken together with the results from sKb(CYT)-GFP and mKb-GFP, it is reasonable to deduce that the CP segment is involved in the association of class I molecules with calnexin and the transmembrane region might play a dynamic role in the dissociation from calnexin. The suggested kinetic association of class I molecules with calnexin is likely to contribute to the different maturation rate between several class I alleles.