Elongation factor 1βγ from Artemia

Abstract

The guanine nucleotide exchange factor, elongation factor 1py (EF-1py) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26000 (EF-1P) and 46000 (EF-ly). A nucleoside diphosphate phosphotransferase activity often found in EF-1py preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF-Ipy, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF-1P. Both EF-lpy and EF-1p have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF-la with exogenous GDP; (b) EF-1cc-dependent binding of Phe-tRNA to ribosomes; (c) poly(U)-dependent poly(phenyla1anine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF-lpy compared to EF-lP alone.Concerning elongation factor l y (EF-ly) the following observations were made. In contrast to EF-Ifl, pure EF-ly is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF-ly partitions solely into the detergent phase of Triton X-114 solutions. EF-1y is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF-lpy was found to be present in membrane fractions, under conditions where no EF-la was detectable in these fractions. Furthermore it was noted that EF-lpy copurified strongly with tubulin on DEAEcellulose. Moreover, it was observed that from a mixture of EF-1py and tubulin, EF-1y coprecipitates with tubulin using a non-denaturating immunoprecipitation technique. These findings suggest that EF-1 y has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.Elongation of polypeptides on ribosomes requires GTP and two protein factors which in eukaryotes are designated as EF-1 and EF-2. Elongation factor EF-1 mediates the binding of aminoacyl-tRNA to the ribosome under hydrolysis of GTP. After peptide-bond formation, translocation of peptidyl-tRNA occurs on the ribosome under influence of elongation factor EF-2 and a second round of GTP hydrolysis. The acidic ribosomal proteins from the large ribosomal subunits are important in this whole process [I]. Elongation factor I is composed of two distinct parts: a nucleotide binding protein, EF-la and a nucleotide exchange protein complex, EF-lpy [2-111. The actual nucleotide exchange activity resides in EF-lB, whereas the function of EF-1y has been unknown so far. For a recent review on elongation factors in the Artemia embryo we refer to [12]. This paper gives a simple method to prepare Artemia EF-lpy and its constituent polypeptides EF-1P and EF-1y in quantities sufficient for structural work. As a result the primary structure of both proteins could be determined [13, 141. We report here some kinetic properties of this EF-1p together with an effect of EF-1 y on th...

A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF‐1γ and EF‐1β

“…from shrimp Artemia salina [9] as depicted in fig.3. Since EF-I~ has been described to form a complex in association with EF-17 [18], we have searched for homology between p47 polypeptides and EF-17 [10].…”

Comparison of phosphorylation of elongation factor 1 from different species by caScin kinase II

“…and Traugh, J.A., manuscript in preparation) and Artemia salina [16]. EF-1 /37(6) was purified from Artemia [5] and wheat germ [9]. The factor from Artemia was generously provied by Dr Wim M611er (State University of Leiden, The Netherlands), and the factor from wheat germ was a gift of Dr Joarine M. Ravel (University of Texas, Austin).…”

“…A significant portion of EF-1 is tightly complexed with valyl-tRNA synthetase [3,4]. EF-1 consists of 3 (c~ fl 3') or 4 (~) subunits as shown with Artemia salina [5], wheat germ [6] and rabbit reticulocytes [7]. The o~-subunit (Mr 50 000) has been isolated alone or in the holoenzyme and mediates the GTP-dependent binding of aminoacyl-tRNA to 80 S ribosomal subunits.…”

“…The o~-subunit (Mr 50 000) has been isolated alone or in the holoenzyme and mediates the GTP-dependent binding of aminoacyl-tRNA to 80 S ribosomal subunits. The f13" subunits have been isolated [5,8,9] and shown to catalyze the exchange of GDP bound to EF-lot with exogenous GTP. An additional subunit, 6, is sometimes found with the f13' complex and with EF-1; the potential role of 6 in elongation has yet to be realized.…”

“…Values were taken from [7] for reticulocytes, from [9] for wheat germ and [5] for Artemia salina. Letters in parentheses denote the nomenclature used in the original paper.…”