EXCEED THE SPACE PROVIDED. Assembly and intracellular trafficking of MHC I molecules is the crucial to their display and antigen presentation to T cells. All of the steps of assembly and peptide loading of MHC I molecules occur in the membrane of one cell organelle, the endoplasmic reticulum, ER. However, the distribution of assembly and quality control regions of the ER remains largely unknown. Biochemistry defines the molecules involved and the temporal dimensions of assembly and quality control, but does not yield their spatial dimensions. It also does not address the steps of ER export after MHC I is peptide loaded. We will ask 3 questions about the ER assembly, quality control and export of MHC I molecules. I. How are MHC I assembly, quality control and carrier-mediated ER export organized over the large area of the ER membrane? Are the individual steps in these processes confined to ER membrane domains or do they occur at random throughout the ER? I1. How does BAP31, an ER export and quality control protein, function in the export and quality control of MHC I molecules? Does it function solely as an export carrier, or is it also an MHC I quality control protein? Ill. Do viral proteins that interfere with ER exit aggregate MHC I molecules and direct them to domains that are isolated from ER exit sites, or do they compete with export carriers for MHC I binding? These questions will be first addressed by a combination of FRAP and FLIP measurements to measure molecular mobility and formation of domains in the ER. Immunoprecipitation and resonance energy transfer, FRET, measurements will detect molecular clustering and molecular associations for ER export and quality control. These techniques will be used to study wild-type and mutant MHC I molecules tagged with GFP and other fluorescent proteins, FP. Chaperones and export carriers, tapasin and BAP31 will also be tagged with FP as will viral proteins, the US3 protein of HCMV, and the MCMV proteins m4(p34) and m152. PERFORMANCE SITE ========================================Section End===========================================

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Allergy and Immunology Study Section (ALY)
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Kirkham, Perry M
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Johns Hopkins University
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