Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain.
Journal Article
GRP94 is an abundant, resident glycoprotein of the mammalian endoplasmic reticulum lumen and member of the hsp90 family of molecular chaperones. To identify the structure/function relationships which define the molecular basis of GRP94 activity, we have performed a structural analysis of native GRP94 and identified a discrete domain, representing amino acids 676-719, which regulates dimerization and displays autonomous oligomerization activity. Velocity sedimentation and gel filtration chromatography were used to identify native GRP94 as a dimer with an extended, rod-like shape. Limited proteolysis resulted in the loss of approximately 16 kDa from the C-terminus and disassembly into monomers, implicating the C-terminus as the site of assembly. An assembly function for the C-terminal domain was established by analysis of the quaternary structure of C-terminal constructs synthesized either in vitro or through recombinant expression. In vitro translation was used to demonstrate that a C-terminal 20 kDa domain was both necessary and sufficient for dimerization. Structural studies of recombinant fusion protein constructs yielded identification of a 44 amino acid domain that displayed autonomous dimerization activity and conferred a highly elongated structure, characteristic of native GRP94, to the fusion protein. These data, combined with molecular dimensions obtained from rotary shadowing electron microscopy, provide a structural model of GRP94 and identify the molecular basis of GRP94 self-assembly.
Full Text
Duke Authors
Cited Authors
- Wearsch, PA; Nicchitta, CV
Published Date
- December 24, 1996
Published In
Volume / Issue
- 35 / 51
Start / End Page
- 16760 - 16769
PubMed ID
- 8988013
Pubmed Central ID
- 8988013
International Standard Serial Number (ISSN)
- 0006-2960
Digital Object Identifier (DOI)
- 10.1021/bi962068q
Language
- eng
Conference Location
- United States