Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration.
There are few reports concerning the sorting mechanisms of mammalian HSP60 into the mitochondria from the cytoplasm. In the present study we investigated the protein import system. Based on immunoblotting and immuno-histochemistry, HSP60 was detected in both the cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay. HSP60 mRNA was increased in the kidney papilla of rats that had been water restricted for three and five days, but no changes in HSP60 mRNA were detected in the cortex or the medulla of the rat kidneys. Upon immunoblotting, HSP60 was detected in both the cytoplasm and the mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the kidney papilla of rats that were water restricted but the protein was increased in the mitochondria of the rat kidney papilla. We also analysed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with a molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from the affinity column by an excess of signal peptide or by 5 mm ATP. Upon immunoblotting, the 70-kDa protein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytoplasmic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP70.
Itoh
Hideaki
H
Department of Biochemistry, Akita University School of Medicine, Akita City, Japan. hideaki@med.akita-u.ac.jp
Komatsuda
Atsushi
A
Ohtani
Hiroshi
H
Wakui
Hideki
H
Imai
Hirokazu
H
Sawada
Ken-Ichi
K
Otaka
Michiro
M
Ogura
Masahito
M
Suzuki
Akira
A
Hamada
Fumio
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D017931:DNA Primers; D003681:Dehydration / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D018834:Chaperonin 60; D017931:DNA Primers; D012333:RNA, Messenger