Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain.

Journal Article (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

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi962068q


  • eng

Conference Location

  • United States