In a process central to the immune response against viruses and tumors, MHC class I molecules present peptide antigens at the cell surface for recognition by CD8+ T cells. MHC class I molecules are loaded with peptide in the endoplasmic reticulum (ER) while sequestered within the peptide loading complex, or PLC. The PLC consists of the Transporter associated with Antigen Processing (TAP), which translocates antigenic peptides into the ER, and an ER-luminal subcomplex. This luminal subcomplex is sufficient for peptide loading as well as for the exchange of low affinity for high affinity peptides, or "peptide editing". In addition to the MHC class I molecule, the subcomplex comprises the lectin chaperone calreticulin, the protein disulfide isomerase ERp57, and tapasin, an MHC class I specific chaperone. How the components of luminal PLC subcomplex cooperate to facilitate peptide loading and editing has been difficult to understand in the absence of a structure for the whole subcomplex. Here we propose to obtain this critical structural information for the entire ER-luminal PLC subcomplex as a means to understanding the molecular mechanisms underlying its function. The first aim is to obtain sufficient material for structure determination. At present, we are able to reconstitute the complex from components in quantities sufficient for single particle electron microscopy (EM), and our challenge is to scale up for crystallographic studies. We are also developing a second approach, where the components of the luminal subcomplex are assembled in insect or human lymphoblastoid cells and the entire subcomplex is then isolated. The second aim is to determine the structure of the ER-luminal PLC subcomplex, either by X-ray crystallography or by EM. Low resolution information from EM will show how tapasin, ERp57, and calreticulin are arranged around the MHC class I molecule, thereby providing clues as to their function in the loading process. An X-ray structure will reveal details regarding how these proteins interact with the MHC class I peptide binding groove and would thus suggest the mechanism by which the PLC facilitates peptide loading and exchange. An understanding of PLC architecture is relevant not only for MHC class I maturation but also more generally for understanding protein folding and quality control in the ER, since ERp57 and calreticulin are general foldases that are not restricted to the MHC class I molecule. PUBLIC HEALTH RELEVANCE: MHC class I molecules present peptide antigens at the cell surface for recognition by the immune system, playing a critical role in the response against viruses and tumors. The class I molecules are loaded with antigenic peptides by the peptide loading complex. We propose to visualize this complex in order to understand the molecular mechanisms by which peptide loading takes place.