Identification of Tetrahymena hsp60 as a 14-nm filament protein/citrate synthase-binding protein and its possible involvement in the oral apparatus formation.
BACKGROUND
Tetrahymena 14-nm filament protein (14FP) is bifunctional, with roles as a citrate synthase in mitochondria and as a cytoskeletal protein in nuclear events during fertilization and in oral morphogenesis. In this study, to further our understanding of the bifunctional property of 14FP, we attempted to screen 14FP-binding proteins using affinity column chromatography.


RESULTS
Through the screening of 14FP-binding proteins using 14FP-affinity chromatography, we detected 65 kDa and 70 kDa proteins that bound to 14FP in an ATP dependent manner. From the N-terminal amino acid sequence, these proteins were identified as the Tetrahymena mitochondrial chaperones, hsp60 and mthsp70, respectively. Tetrahymena hsp60 was recognized with a monoclonal antibody raised against human hsp60. Immunofluorescence and immunoelectron microscopy using the monoclonal antibody showed that Tetrahymena hsp60 was localized to mitochondria. Moreover, Tetrahymena hsp60 was also present at extramitochondrial sites including basal bodies of cilia and oral apparatus, and particularly at the developing oral apparatus during cell division.


CONCLUSIONS
These results suggest that Tetrahymena hsp60 is localized in basal bodies and is involved in cortical patterning such as the formation of the oral apparatus as well as having a role in the folding of mitochondrial proteins in mitochondria.
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572 Japan.
T
T
Takeda
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I
Yoshihama
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O
Numata
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002455:Cell Division; D018834:Chaperonin 60 / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013768:Tetrahymena / Q000378:metabolism*
D018834:Chaperonin 60; C430588:hsp60 protein, Tetrahymena; D002950:Citrate (si)-Synthase