Intracellular transport of proteins containing di-arginine endoplasmic reticulum-retention motifs

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RATIONALE The intracellular trafficking of MHC class II molecules (MHCIIs) represents a particular challenge as they must traverse many different compartments and interact with various proteins to finally fulfil their functions at the plasma membrane. The invariant chain (CD74; Ii) chaperone assists MHCIIs in their folding and trafficking to the endocytic pathway. In humans, two Ii isoforms display a di-arginine motif (RxR) on their cytoplasmic tail. The RxR sequence acts as an endoplasmic reticulum (ER) retention motif and it has been shown to play a key role in protein quality control. The role of Ii's RxR motif remains nebulous and the mechanism by which it is "masked" by MHCIIs to allow ER egress is ill-defined. Thus, we propose to further our understanding of the basics of protein trafficking across sub-cellular compartments and to document the protein networks involved in the functions of Ii and MHCIIs. Recent Progress We adapted a new proximity ligation assays-dependent biotin identification (BioID) assay to identify protein-protein interactions involved in the intracellular trafficking of MHCIIs and Ii. To this end, we have used the CRISPR/Cas genome engineering technology in B lymphoblastic cell lines to inactivate the Ii gene. Our results demonstrate that the scaffolding role of Ii on the association of MHC class II molecules is allotype-dependent. These Ii-deficient B cell lines allow us to reconstitute the exogenous antigen presentation pathway with various isoforms and mutants of Ii and/or MHCIIs. SPECIFIC AIM 1: To identify molecules regulating MHCII intracellular transport. We will identify the proteins that across the path of MHCIIs in B cells and epithelial cells in the absence of the Ii chaperone. SPECIFIC AIM 2: To characterize the interplay between Ii and MHCIIs in the transport of the complex. We will reconstitute Ii-negative cells with the different isoforms of Ii and study the specific interactions taking place with the RxR ER retention motif. SPECIFIC AIM 3: To characterize the function of key regulators of MHCII/Ii trafficking and antigen processing. We will validate the importance of the interactors identified in aims 1 and 2 by modulating the expression (mRNA knockdown or cDNA overexpression) of the proteins in B cells or epithelial cells expressing MHCIIs and Ii. The impact on the presentation of antigens to T cells will be assessed. METHODOLOGY: We will use a combination of molecular biology (site-directed mutagenesis, proximity ligation assays), cell biology (confocal microscopy and flow cytometry) and biochemistry (Western blotting, protein purification and mass spectrometry) techniques to dissect and map the interactions occurring between Ii isoforms, MHCIIs and their partners. IMPACT: Our results will further our understanding of protein sorting in general and will give clues as to the role of Ii's cytoplasmic motifs in its own intracellular trafficking and in controlling the fate of MHCIIs.