TY - JOUR
T1 - An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells
AU - Ostrovsky, Olga
AU - Makarewich, Catherine A.
AU - Snapp, Erik L.
AU - Argon, Yair
PY - 2009/7/14
Y1 - 2009/7/14
N2 - 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.
AB - 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.
KW - Endoplasmic reticulum
KW - Peptide hormones
KW - Protein folding
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U2 - 10.1073/pnas.0902626106
DO - 10.1073/pnas.0902626106
M3 - Article
C2 - 19553200
AN - SCOPUS:67650882756
SN - 0027-8424
VL - 106
SP - 11600
EP - 11605
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 28
ER -