Peptide-receptive Major Histocompatibility Complex Class I Molecules Cycle between Endoplasmic Reticulum and cis-Golgi in Wild-type Lymphocytes

Abstract

Prior to binding to a high affinity peptide and transporting it to the cell surface, major histocompatibility complex class I molecules are retained inside the cell by retention in the endoplasmic reticulum (ER), recycling through the ER-Golgi intermediate compartment and possibly the cis-Golgi, or both. Using fluorescence microscopy and a novel in vitro COPII (ER-to-ERGolgi intermediate compartment) vesicle formation assay, we find that in both lymphocytes and fibroblasts that lack the functional transporter associated with antigen presentation, class I molecules exit the ER and reach the cis-Golgi. Intriguingly, in wild-type T1 lymphoma cells, peptide-occupied and peptidereceptive class I molecules are simultaneously exported from ER membranes with similar efficiencies. Our results suggest that binding of high affinity peptide and exit from the ER are not coupled, that the major histocompatibility complex class I quality control compartment extends into the Golgi apparatus under standard conditions, and that peptide loading onto class I molecules may occur in post-ER compartments.

Peptide-independent stabilization of MHC class I molecules breaches cellular quality control*

“…When we co-expressed b 2 m with the K b heavy chains, we observed that wild-type K b now exited the ER but accumulated in the p58-positive compartment (Fig. 7E, bottom left, arrows), indicating that it was retained there by the ERGIC and cis-Golgi quality control (Garstka et al, 2007). Small amounts were detected at the cell surface.…”

“…Radiolabeling and immunoprecipitation were performed as described previously (Fritzsche and Springer, 2013). Immunofluorescence microscopy was performed as described previously (Garstka et al, 2007).…”

“…MHC-I molecules are again monitored for high-affinity peptide occupancy by a system that is related to the standard glycoprotein quality control (Ellgaard and Helenius, 2001). It recognizes suboptimally loaded MHC-I, most likely through UDP-glucose:glycoprotein glucosyltransferase (UGT1), retains them in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, prevents them from becoming endoglycosidase H resistant, and eventually returns them to the ER with the help of calreticulin (Hsu et al, 1991;Paulsson et al, 2006;Garstka et al, 2007;Purcell and Elliott, 2008;Wearsch and Cresswell, 2008;Howe et al, 2009;Zhang et al, 2011). This second system is responsible for the intracellular retention of tapasin-dependent MHC-I allotypes in tapasin-deficient cells (Peh et al, 1998;Garstka et al, 2011).…”

Tapasin-related protein TAPBPR is an additional component of the MHC class I presentation pathway

“…Post-PLC, MHC I molecules are exported from the ER via a selective process, involving trafficking motifs in the HC cytoplasmic tails and Bap31, which facilitates recruitment into COP-II coated vesicles (31)(32)(33)(34)(35)(36). A bottleneck in the MHC I pathway appears to occur before the medial Golgi with the transport of suboptimally loaded MHC I molecules and PLC components blocked at this step (4,(37)(38)(39)(40). Our most intriguing finding is the ability of TAPBPR to stay associated with MHC I beyond the ER/cis-Golgi, a function that clearly sets TAPBPR apart from all other known components of the MHC I presentation pathway.…”

Tapasin edits peptides on MHC class I molecules by accelerating peptide exchange

“…In cell-based assays, tapasin leads to the increased surface expression, thermostability, and persistence of class I-peptide complexes [4,[7][8][9], but some reports state that it does not influence the composition of the peptide pool bound to class I [10,11]. In assays that use whole cells, the peptide editing function of tapasin may be obscured by ER/Golgi quality control [12], a role of tapasin in the trafficking of class I to or from the Golgi apparatus (Springer et al, unpublished data) [13,14], class I loading by alternative pathways [2], and the metabolic stabilization of TAP by tapasin [3]. In search of the molecular mechanism of peptide editing, several groups have therefore established in vitro assays for tapasin function.…”