This proposal is designed to explore the role of immunoglobulin heavy chain binding protein(BiP) in regulating protein transport through the secretory pathway. We will use immunoglobulin (ig) synthesis as a model system for monitoring B1) association with secretory pathway proteins. Initial experiments will determine whether proteins that combine with BiP can be released and continue along the secretory pathway of whether the binding of BiP to proteins ins irreversible and signals their intracellular degradation. The site of degradation of proteins that remain bound to BiP and the role of Bip, if any, in their degradation will be determined. The role of post-translational modifications in regulating the function of BiP will be explored. Initial experiments indicate that two modifications of BiP, phosphorylation and ADP- ribosylation, are differentially segregated between free and bound pools of BiP and that these pools may be interconvertible. Therefore, we will focus on these modifications in the association, dissociation and degradation of ig heavy chains. finally, we will attempt to produce BiP conditional cellular mutants to more directly assess the role of BiP in monitoring protein transport along the secretory pathway. The experiments described in this proposal will use a combination of biochemical and genetic approaches to elucidate the function of BiP.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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St. Jude Children's Research Hospital
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Hendershot, L; Wei, J; Gaut, J et al. (1996) Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants. Proc Natl Acad Sci U S A 93:5269-74
Hendershot, L M; Wei, J Y; Gaut, J R et al. (1995) In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum. Mol Biol Cell 6:283-96
Wei, J; Gaut, J R; Hendershot, L M (1995) In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis. J Biol Chem 270:26677-82
Wei, J; Hendershot, L M (1995) Characterization of the nucleotide binding properties and ATPase activity of recombinant hamster BiP purified from bacteria. J Biol Chem 270:26670-6
Fitts, M G; Metzger, D W; Hendershot, L M et al. (1995) The rabbit B cell antigen receptor is non-covalently associated with unique heteromeric protein complexes: possible insights into the membrane IgM/IgD coexpression paradox. Mol Immunol 32:753-9
Hendershot, L M; Valentine, V A; Lee, A S et al. (1994) Localization of the gene encoding human BiP/GRP78, the endoplasmic reticulum cognate of the HSP70 family, to chromosome 9q34. Genomics 20:281-4
Gaut, J R; Hendershot, L M (1993) The immunoglobulin-binding protein in vitro autophosphorylation site maps to a threonine within the ATP binding cleft but is not a detectable site of in vivo phosphorylation. J Biol Chem 268:12691-8
Gaut, J R; Hendershot, L M (1993) Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain. J Biol Chem 268:7248-55
Freiden, P J; Gaut, J R; Hendershot, L M (1992) Interconversion of three differentially modified and assembled forms of BiP. EMBO J 11:63-70