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

The molecular event governing the assembly of the MHC class I heavy chain-b eta 2-microglobulinpeptide complex is still not fully understood, In order to characterize the transport properties of MHC class I molecules, several truncated H-2K(b) genes were constructed and expressed in COS7 cells. Surpr isingly, the expressed soluble molecule containing connecting peptide (CP) segment (sK(CP)(b)) did not secrete as efficiently as the one without CP (s K(CYT)(b)). When the sK(b)(CP) gene was transfected into a calnexin-deficie nt cell line CEM.NKR, the amount of soluble K-b molecules in the supernatan t was comparable with sK(CYT)(b)-transfected CEM.NKR, To further demonstrat e the different transport of sK(CP)(b) and sK(CYT)(b) within living cells, we attached green fluorescent protein (GFP) to the C-termini of both molecu les and, as a comparison, to the full-length transmembrane counterpart (mK( b)-GFP). While the mK(b)-GFP-transfected cells showed the green fluorescenc e in the reticular network and the nuclear envelope, sK(CP)(b)-GFP showed o bviously lump fluorescence of high intensity within cells, However, the dis tribution of sK(CYT)(b)-GFP was fairly uniform, Furthermore, GFP-tagged mol ecules allow us to analyze their interaction with other proteins in a direc t, simple and quantitative method, designated immunofluorescence precipitat ion, The results showed that 60% of sK(CP)(b)-GFP molecules were associated with calnexin, while <10% with tapasin, Taken together with the results fr om sK(CYT)(b)-GFP and mK(b)-GFP, it is reasonable to deduce that the CP seg ment is involved in the association of class 1 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 caln exin is likely to contribute to the different maturation rate between sever al class 1 alleles.