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

Publication ,  Journal Article
Wearsch, PA; Nicchitta, CV
Published in: Biochemistry
December 24, 1996

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.

Duke Scholars

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

December 24, 1996

Volume

35

Issue

51

Start / End Page

16760 / 16769

Location

United States

Related Subject Headings

  • Swine
  • Recombinant Fusion Proteins
  • Protein Conformation
  • Molecular Structure
  • Molecular Chaperones
  • Membrane Proteins
  • HSP70 Heat-Shock Proteins
  • Endoplasmic Reticulum
  • Dimerization
  • Detergents
 

Citation

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Wearsch, P. A., & Nicchitta, C. V. (1996). Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain. Biochemistry, 35(51), 16760–16769. https://doi.org/10.1021/bi962068q
Wearsch, P. A., and C. V. Nicchitta. “Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain.Biochemistry 35, no. 51 (December 24, 1996): 16760–69. https://doi.org/10.1021/bi962068q.
Wearsch, P. A., and C. V. Nicchitta. “Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain.Biochemistry, vol. 35, no. 51, Dec. 1996, pp. 16760–69. Pubmed, doi:10.1021/bi962068q.
Wearsch PA, Nicchitta CV. Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain. Biochemistry. 1996 Dec 24;35(51):16760–16769.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

December 24, 1996

Volume

35

Issue

51

Start / End Page

16760 / 16769

Location

United States

Related Subject Headings

  • Swine
  • Recombinant Fusion Proteins
  • Protein Conformation
  • Molecular Structure
  • Molecular Chaperones
  • Membrane Proteins
  • HSP70 Heat-Shock Proteins
  • Endoplasmic Reticulum
  • Dimerization
  • Detergents