An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells

Olga Ostrovsky, Catherine A. Makarewich, Erik L. Snapp, Yair Argon

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone for which only few client proteins and no cofactors are known and whose mode of action is unclear. To decipher the mode of GRP94 action in vivo, we exploited our finding that GRP94 is necessary for the production of insulin-like growth factor (IGF)-II and developed a cell-based functional assay. Grp94-/- cells are hypersensitive to serum withdrawal and die. This phenotype can be complemented either with exogenous IGF-II or by expression of functional GRP94. Fusion proteins of GRP94 with monomeric GFP (mGFP) or mCherry also rescue the viability of transiently transfected, GRP94-deficient cells, demonstrating that the fusion proteins are functional. Because these constructs enable direct visualization of chaperone-expressing cells, we used this survival assay to assess the activities of GRP94 mutants that are defective in specific biochemical functions in vitro. Mutations that abolish binding of adenosine nucleotides cannot support growth in serumfree medium. Similarly, mutations of residues needed for ATP hydrolysis also render GRP94 partially or completely nonfunctional. In contrast, an N-terminal domain mutant that cannot bind peptides still supports cell survival. Thus the peptide binding activity in vitro can be uncoupled from the chaperone activity toward IGF in vivo. This mutational analysis suggests that the ATPase activity of GRP94 is essential for chaperone activity in vivo and that the essential protein-binding domain of GRP94 is distinct from the N-terminal domain.

Original languageEnglish (US)
Pages (from-to)11600-11605
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number28
DOIs
StatePublished - Jul 14 2009

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Hydrolysis
Adenosine Triphosphate
Insulin-Like Growth Factor II
Cell Survival
glucose-regulated proteins
Peptides
Mutation
Proteins
Cell Fusion
Somatomedins
Protein Binding
Endoplasmic Reticulum
Adenosine
Adenosine Triphosphatases
Nucleotides
Phenotype
Growth
Serum

Keywords

  • Endoplasmic reticulum
  • Peptide hormones
  • Protein folding

ASJC Scopus subject areas

  • General

Cite this

An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells. / Ostrovsky, Olga; Makarewich, Catherine A.; Snapp, Erik L.; Argon, Yair.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 28, 14.07.2009, p. 11600-11605.

Research output: Contribution to journalArticle

Ostrovsky, Olga ; Makarewich, Catherine A. ; Snapp, Erik L. ; Argon, Yair. / An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2009 ; Vol. 106, No. 28. pp. 11600-11605.
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