Efficient endosomal localization of major histocompatibility complex class II-invariant chain complexes requires multimerization of the invariant chain targeting sequence.

Abstract

Abstract. During biosynthesis, MHC class II-invariant chain complexes are transported into endosomal compartments where invariant chain (Ii) is degraded and class II encounters antigenic peptides. One of the signals that determines this intracellular transport route has been localized to the cytosolic domain of Ii. Deletion of this signal disrupts endosomal targeting and resuits in the stable expression of class II-Ii complexes at the surface. In this paper we have examined the role of Ii trimerization on the generation of this endosomal localization signal. In L cell transfectants expressing class II and both wild type Ii and a truncated form of Ii that lacks this endosomal localization signal, Ii was found to form multimers which could contain both wild type and truncated Ii. The multimers were not large aggregates but were found to be discrete complexes, probably the nine molecule class II-Ii complex that has been observed in human B cells. The co-expression of truncared Ii allowed for cell surface expression of a subset of wild type Ii. This surface-expressed wild type Ii associated with truncated Ii in multimers at a 2:1 ratio, indicating that these trimers contain two truncated and one wild type Ii molecule. These data suggest a division in trafficking of Ii trimers: if two wild type Ii molecules are present, the complex is transported to and rapidly degraded in endosomes, whereas the presence of only one wild type Ii results in trafficking and expression of the heterotrimer on the cell surface. Following surface arrival, complexes containing only a single wild type Ii molecule are internalized more rapidly and have a shorter half-life than complexes containing only truncated Ii molecules. These data suggest that although a single Ii cytosolic domain can function as a plasma membrane internalization signal, multimerization of Ii is required for efficient Golgi complex to endosome targeting of class II-Ii complexes.

The chondroitin sulfate form of invariant chain trimerizes with conventional invariant chain and these complexes are rapidly transported from thetrans-Golgi network to the cell surface

“…Alternatively, some proteoglycans are degraded in endocytic compartments following internalization [35,36]. The cytosolic domain of Ii functions as an efficient internalization signal [20,37,38]. Therefore rapid endocytosis of Ii-CS from the cell surface and lysosomal degradation could account for the short half-life of Ii-CS.…”

“…Cells were metabolically labelled with either [ 3 H]leucine or 35 SO 4 as described in [11,20]. Cells (2 × 10 6 cells/ml) were labelled with [ 35 S]methionine at a concentration of 200 µCi/ml or [ 3 H]leucine at 300 µCi/ml.…”

“…Labelled cells were lysed and pre-cleared, and lysates were immunoprecipitated with the indicated antibody as described previously [20]. Immunoprecipitated protein was eluted, boiled and then separated by SDS/PAGE (10% gels).…”

“…Immunoprecipitated protein was eluted, boiled and then separated by SDS/PAGE (10% gels). Gels were then treated with En 3 Hance TM (New England Nuclear), dried and radiographed as described previously [20]. Immunoprecipitated protein was treated with endoglycosidase H and N-glycosidase (Calbiochem) as described previously [27].…”

“…Alternatively, the bulk of MHCII-Ii complexes may be transported directly from the TGN to endosomes, while a minority of MHCIIIi may be diverted to the plasma membrane [19]. We have shown previously that multimerization of the cytosolic tail of Ii is required for efficient transport of MHCII-Ii complexes to endosomes [20]. Ii trimers that contained only a single intact cytosolic tail were enriched at the cell surface, suggesting that regulation of Ii trimerization may contribute to the cell-surface pool of Ii.…”