The normal development and function of the immune system is dependent, in part, on the proper assembly and expression of immunoglobulin (Ig) receptor and secreted proteins. Like all secretory pathway proteins, Ig molecules are synthesized, folded and assembled in the endoplasmic reticulum (ER), but unlike most heteromeric proteins, there is a developmental asymmetry in the expression of the subunits. The molecular chaperones of the ER play a critical role in controlling the folding of the individual subunits and ensuring that incompletely or improperly assembled Ig proteins do not leave this organelle. Therefore, elucidating the mechanisms by which Ig maturation in the ER is controlled is important to understanding how the proper development of the immune system is ensured. In the past, Ig heavy and light chains have also provided an excellent model system for identifying components of the ER quality control system and for understanding their function. During the last funding period, our studies revealed that Ig transport is controlled at the level of domain folding and that unlike other unfolded proteins, unassembled heavy chains do not appear to cycle on and off ER chaperones. To understand the cellular machinery that is responsible for this, experiments are proposed in the present application to 1) determine the role of GRP94 (a major ER chaperone of unknown function) in maintaining heavy chains in an unfolded state that is competent for assembly with light chains, 2) identify and characterize ER proteins that regulate the ATPase cycle of BiP, an ER chaperone, in order to understand why it remains stably bound to Ig heavy chains in the absence of light chain synthesis, and 3) isolate ER homologues of the DnaJ chaperone cofactor, which acts to aid and stabilize the binding of hsp70 chaperones to unfolded proteins to understand how BiP's binding to heavy chains is controlled. The data obtained from the experiments outlined in this proposal will increase our understanding of the ER quality control machinery that is so crucial to proper Ig biosynthesis and maturation.
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