| Research Abstract |
Tetrahymena 14-nm filament protein/citrate synthase (14FP) is a bifunctional protein with roles in the cytoskeleton and as a citrate synthase. In this study, I examined regulation systems of bifunctional properties of 14FP.
(1) A recombinant 14FP was expressed in E. coli, purified and characterized. The recombinant 14FP has the citrate synthase activity and formed 14-nm filaments. These results suggest that 14FP itself is bifunctional.
(2) Analysis by two-dimensional gel electrophoresis demonstrated that the citrate synthase purified from Tetrahymena mitochondria comprised pI. 7.7 and 8.0 isoforms, while the 14-nm filament protein purified by the assembly and disassembly procedures comprised pI. 7.7, 8.0 and 8.4 isoforms. The pI. 7.7 and/or pI. 8.0 isoforms exhibit the citrate synthase activity, but the pI. 8.4 isoform did not possess this activity. These results suggest that the polymorphism of these isoforms is caused by some posttranslational modifications.
(3) All isoforms were phosph
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orylated, and polymorphism of its isoforms resulted from differences in the degree of phosphorylation between them. Moreover, dephosphorylation led to a decrease in citrate synthase activity and activated filament-forming ability of 14FP. These results suggest that phosphorylation and dephosphorylation regulate the bifunctional properties of 14FP.
(4) The screening of 14FP-binding proteins using 14FP-affinity chromatography demonstrated that Tetrahymena mitochondrial chaperones, hsp60 and mthsp70, bound to 14FP in an ATP dependent manner. Immunofluorescence microscopy using the monoclonal antibody showed that Tetrahymena hsp60 was localized to mitochondria, possibly participating in the folding of mitochondrial proteins including 14FP. Moreover, Tetrahymena hsp60 was also present at extramitochondrial sites including basal bodies and oral apparatus, especially at developing oral apparatus during cell division. Since 14FP participates in the oral morphogenesis, it is possible that Tetrahymena hsp60 is involved in the oral morphogenesis and regulates the bifunctional properties of 14FP. Less
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