Yasuhiro Kurasawa

Identification of Tetrahymena 14-nm filament-associated protein as elongation factor 1α

Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during conjugation. By immunoblotting using monoclonal and polyclonal antibodies following two-dimensional gel electrophoresis, we demonstrated that the 14-nm filament protein fraction contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0; a monoclonal antibody (MAb) 26B4 and a polyclonal antibody 49KI reacted only to a pI 8.0 protein, while two other MAbs, 11B6 and 11B8, reacted only to a pI 9.0 protein. From the N-terminal amino acid sequences, the pI 8.0 protein was identified as the previously reported 14-nm filament-forming protein/citrate synthase, but the pI 9.0 protein N-terminal sequence had no similarity with that of the pI 8.0 protein. The pI 9.0 protein is considered to be a 14-nm filament-associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a Tetrahymena pyriformis cDNA library, we identified the pI 9.0 protein as elongation factor 1 alpha (EF-1 alpha) based on it sharing 73-76% sequence identity with EF-1 alpha from several species.

Characterization of F-Actin Bundling Activity of Tetrahymena Elongation Factor 1α Investigated with Rabbit Skeletal Muscle Actin

Abstract Elongation factor 1&agr; (EF-1&agr;) is an essential factor for protein synthesis in eukaryotes. Here, we demonstrated that Tetrahymena EF-1&agr; induced bundles of rabbit skeletal muscle F-actin as well as Tetrahymena F-actin in vitro, although Tetrahymena and skeletal muscle actins are different in some parts of their primary structures and in the binding abilities to some actin-binding proteins. Co-sedimentation experiments showed that the binding ratio of Tetrahymena EF-1&agr; to skeletal muscle F-actin in the bundles was 1:1 . Electron microscopic observation showed that alkaline pH or high ionic strength reduced the bundling activity of Tetrahymena EF-1&agr; to some extent, although the EF-1&agr; seemed to be able to induce bundling of the F-actin within the range of physiological condition.

Cytokinesis in Tetrahymena: Determination of division plane and organization of contractile ring

A protein, Tetrahymena p85, is localized to the presumptive division plane before the formation of the contractile ring. p85 directly interacts with Tetrahymena calmodulin (CaM) in a Ca2+‐dependent manner, and p85 and CaM colocalize in the division furrow. A Ca2+/CaM inhibitor N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide HCl (W7) inhibits the direct interaction between p85 and Ca2+/CaM. W7 also inhibits the localization of p85 and CaM to the division plane, and the formation of the contractile ring and division furrow. Tetrahymena fimbrin and elongation factor‐1a (EF‐1α), which induce bundling of Tetrahymena F‐actin, are also localized to the division furrow during cytokinesis. The Tetrahymena fimbrin has two actin‐binding domains, but lacks the EF‐hand Ca2+‐binding motif, suggesting that Tetrahymena fimbrin probably cross‐links actin filaments in a Ca2+‐ insensitive manner during cytokinesis. The evidence also indicates that Ca2+/CaM inhibits the F‐actin‐bundling activity of EF‐1α; and EF‐1α and CaM colocalize in the division furrow. In this review, we propose that the Ca2+/CaM signal and its target protein p85 cooperatively regulate the determination of the division plane, and that a Ca2+‐insensitive actin‐bundling protein, Tetrahymena fimbrin, and a Ca2+/CaM‐sensitive actin‐bundling protein, EF‐1α, play pivotal roles in regulating the organization of the contractile ring microfilaments. Microsc. Res. Tech. 49:127–135, 2000.

Antibodies against Tetrahymena 14-nm filament-forming protein recognize the replication band in Euplotes☆

Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein which is involved in activity of the pronuclei during conjugation (O. Numata, T. Sugai, and Y. Watanabe (1985) Nature (London) 314, 192-194). Using monoclonal and polyclonal antibodies, we here demonstrate the presence of a cross-reactive protein (CRP-49) within the macronuclear replication bands of Euplotes harpa and E. eurystomus which is recognized by anti-49K protein antibodies. Immunoblotting reveals that both monoclonal and polyclonal antibodies cross-react to a protein with an apparent molecular mass of 50 kDa in an E. harpa cell extract and to a protein of 49 kDa in a macronuclear extract of E. eurystomus. The antibodies used in this study have no effect upon in vitro DNA synthesis in the replication band of E. eurystomus.

Tetrahymena Elongation Factor-1α Binds to Hsp70 Family Proteins

Abstract Translation elongation factor 1α (EF-1α) catalyzes the GTP-dependent binding of aminoacyl-tRNA to the ribosome. We previously reported that Tetrahymena EF-1α induced bundles of rabbit skeletal muscle F-actin as well as Tetrahymena F-actin (Kurasawa et al., (1996) Zool. Sci. 13: 371–375), and that Ca2+/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1α without inhibition of the binding between EF-1α and F-actin (Kurasawa et al., (1996) J. Biochem. 119: 791–798). In this study, we investigated EF-1α-binding proteins in Tetrahymena using a Tetrahymena EF-1α affinity column. Tetrahymena EF-1α bound directly to 74 kDa, 77 kDa, and 78 kDa proteins, in addition to CaM. The bindings of 74 kDa, 77 kDa, and 78 kDa proteins to Tetrahymena EF-1α were Ca2+-independent and ATP-sensitive. The N-terminal amino acid sequence of the 74 kDa protein was similar to those of 70 kDa heat shock protein (hsp70) family.