A critical role for the proteasome activator PA28 in the Hsp90-dependent protein refolding.
The 90-kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation and prevent it from undergoing an irreversible off-pathway aggregation, thereby maintaining it in a folding-competent state. While Hsp90 by itself was not sufficient to refold the denatured luciferase, addition of rabbit reticulocyte lysate remarkably restored the luciferase activity. Here we demonstrate that Hsc70, Hsp40, and the 20 S proteasome activator PA28 are the effective components in reticulocyte lysate. Purified Hsc70, Hsp40, and PA28 were necessary and sufficient to fully reconstitute Hsp90-initiated refolding. Kinetics of substrate binding support the idea that PA28 acts as the molecular link between the Hsp90-dependent capture of unfolded proteins and the Hsc70- and ATP-dependent refolding process.
Minami
Y
Y
Department of Biochemistry, Oita Medical University, 1-1 Idaigaoka, Hasama-machi, Oita 879-5593, Japan. minami@oita-med.ac.jp
Kawasaki
H
H
Minami
M
M
Tanahashi
N
N
Tanaka
K
K
Yahara
I
I
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D003546:Cysteine Endopeptidases; D004789:Enzyme Activation; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006358:Hot Temperature; D008156:Luciferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D011506:Proteins; D008156:Luciferases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex