Prominence of beta 2-microglobulin, class I heavy chain conformation, and tapasin in the interactions of class I heavy chain with calreticulin and the transporter associated with antigen processing.

Abstract

Newly synthesized class I heavy (H) chain/beta 2m heterodimers awaiting peptides in the endoplasmic reticulum are associated with the transporter associated with Ag processing (TAP). We present evidence here that calreticulin, but not calnexin, displays steady state association with class I/TAP complexes. To separate the ability of beta 2m to bind with TAP and calreticulin from that of H chain, we studied human cell lines that lack expression of beta 2m or H chain. Little if any H chain was detected in association with TAP and calreticulin in the beta 2m- cell line Daudi. By contrast, high levels of beta 2m are found with TAP and calreticulin in the H chain-deficient cell line LCL 721.221, even after preclearance of the trace amount of class IB protein expressed by this cell line. Thus, beta 2m appears to bind TAP in the absence of H chain, providing an elegant mechanism to retain beta 2m in the endoplasmic reticulum at the site of peptide loading. To investigate whether other molecules participate in the binding of beta 2m and H chain to TAP and calreticulin, we analyzed the deletion mutant cell line LCL 721.220, which lacks tapasin. In 721.220, TAP and calreticulin are not associated with each other. Also, in these cells, H chain/beta 2m are not associated with TAP, but H chain and a low level of beta 2m are associated with calreticulin. These results suggest that tapasin is an obligatory mediator of the assemblage of calreticulin/H chain/beta 2m with TAP.

Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

“…An interaction between calreticulin and sub‐optimally loaded class I molecules outside the PLC is conceivable, since calreticulin needs no co‐factor to recognize its substrates (Peterson and Helenius, 1999), and since it was detected in a complex with class I in TAP‐deficient cells (Sadasivan et al , 1996). Indeed, calreticulin binds to class I in the absence of tapasin (Solheim et al , 1997; Turnquist et al , 2002), and a number of mutant forms of H‐2D d , which do not bind tapasin, were shown to maintain a robust association with calreticulin (Paquet and Williams, 2002). Furthermore, the intracellular retention of sub‐optimally loaded class I is known to be independent of the PLC: first, in tapasin‐deficient cells (where no association of class I with the PLC is possible), tapasin‐dependent class I molecules do not reach the cell surface (Lauvau et al , 1999; Purcell et al , 2001; Garstka et al , in preparation); and second, T134K mutants of class I molecules (which do not associate with tapasin) are still dependent on the peptide transporter, TAP, for surface expression, and are thus subject to normal quality control (Lewis et al , 1996; Sadasivan et al , 1996).…”

Atlantic cod (Gadus morhua) MHC I localizes to endolysosomal compartments independently of cytosolic sorting signals

“…Furthermore, Atlantic cod MHC I variant 1 and tapasin variant 1 interacts and colocalize on endolysosomes ( Figures 6B, D–F ). While the interaction between tapasin and MHC I in Atlantic cod is not unexpected, as it also occurs in mammals ( Rizvi and Raghavan, 2010 ; Blees et al, 2017 ), it is surprising that in Atlantic cod tapasin localizes to the endolysosomal system rather than to the ER as in mammals ( Sadasivan et al, 1996 ; Solheim et al, 1997 ; Rizvi and Raghavan, 2010 ). This is intriguing as it might indicate that the function of Atlantic cod tapasin changed or followed a different evolutionary path.…”