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

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi962068q

Language

  • eng

Conference Location

  • United States