Wim Möller

Elongation factor 1βγ from Artemia

The guanine nucleotide exchange factor, elongation factor 1 beta gamma (EF-1 beta gamma) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26,000 (EF-1 beta) and 46,000 (EF-1 gamma). A nucleoside diphosphate phosphotransferase activity often found in EF-1 beta gamma preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF-1 beta gamma, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF-1 beta. Both EF-1 beta gamma and EF-1 beta have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF-1 alpha with exogenous GDP; (b) EF-1 alpha-dependent binding of Phe-tRNA to ribosomes; (c) poly(U)-dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF-1 beta gamma compared to EF-1 beta alone. Concerning elongation factor 1 gamma (EF-1 gamma) the following observations were made. In contrast to EF-1 beta, pure EF-1 gamma is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF-1 gamma partitions solely into the detergent phase of Triton X-114 solutions. EF-1 gamma is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF-1 beta gamma was found to be present in membrane fractions, under conditions where no EF-1 alpha was detectable in these fractions. Furthermore it was noted that EF-1 beta gamma copurified strongly with tubulin on DEAE-cellulose. Moreover, it was observed that from a mixture of EF-1 beta gamma and tubulin, EF-1 gamma coprecipitates with tubulin using a non-denaturating immunoprecipitation technique. These findings suggest that EF-1 gamma has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.

Elongation factor 1β of artemia: Localization of functional sites and homology to elongation factor 1δ

Elongation factor (EF)-1β, a 26 kDa protein, is the eukaryotic equivalent of bacterial EF-Ts, the nucleotide exchange factor in protein synthesis. EF-1β catalyzes the exchange of guanine nucleotides bound to EF-1α; the latter protein is the eukaryotic equivalent of bacterial EF-Tu. Limited proteolytic cleavage studies on EF-1β lead to the following picture: the protein is composed of two domains, an aminoterminal and a carboxyterminal domain, connected to each other by a stretch of hydrophilic, charged amino acids situated in the middle of the molecule. The carboxyterminal domain supplies the catalytic site for the nucleotide exchange reaction, whereas the aminoterminal domain interacts with EF-1γ, the third component of elongation factor 1. The regulatory, serine phosphate residue, Ser-89, localized in the hydrophilic stretch of EF-1β, does not appear to be necessary for the basic exchange reaction. The fourth component of the high molecular weight elongation factor complex (EF-1H), named EF-1δ or 28 K protein, is homologous to EF-1β and contains regions very similar to the carboxyterminal part. EF-1δ was found to be active in the nucleotide exchange reaction.

A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance.

We have determined the primary structure of a mutant insulin receptor of a leprechaun patient born from a consanguineous marriage. A characteristic feature of leprechaunism is an extreme resistance to insulin. In this patient the insulin resistance seems to result from an observed lack of insulin binding to intact cells. Solubilization of cells in non‐ionic detergents leads to the appearance of insulin receptors which can bind insulin. However, the insulin‐stimulated autophosphorylation of the receptors beta subunit is markedly reduced. Cloning and sequencing of cDNA derived from insulin receptor mRNA of this patient revealed a leucine‐to‐proline mutation at position 233 in the alpha subunit. By means of DNA amplification we found that the patient is homozygous for this mutation and that the parents and two grandparents from the consanguineous line are heterozygous. The heterozygous individuals all show decreased insulin binding to cultured fibroblasts. In addition, they are mildly insulin resistant in vivo. These observations show a linkage between the leucine‐to‐proline mutation and the observed insulin resistance in this family. We therefore conclude that the mutation in the homozygous form is responsible for the extreme insulin resistance in the leprechaun patient. The mutation for the first time characterizes a region in the insulin receptor which seems to be involved in transmitting the insulin binding signal to the tyrosine kinase domain.

The primary structure of elongation factor EF-1 alpha from the brine shrimp Artemia.

cDNA as well as amino acid sequencing has revealed the complete primary structure of elongation factor EF‐1 alpha from the brine shrimp Artemia. A comparison with the published sequences of bacterial EF‐Tu, mitochondrial EF‐Tu and chloroplastic EF‐Tu shows that distinct areas of these polypeptide chains are conserved in evolution. The evolutionary distance between prokaryotic and eukaryotic types of EF‐Tu is larger than among bacterial and organellar EF‐ Tus . A number of regions present in both EF‐Tu and EF‐G from Escherichia coli are also found in EF‐1 alpha from Artemia.

Characterization of an acidic protein in 50 s ribosomes of E. coli

The presence of an alanine rich, acidic protein, called ribosomal A-protein, has been reported in 70 S ribosomes of E. coli [ 1,2] . In this communication, evidence is presented that this protein is an integral part of the 50 S ribosome and is estimated to contain 1wo acidic peptide chains of molecular weight 19,000. The possibility that A-protein is involved in peptide bond formation [3] is discussed. 2. Materials and methods 70 S Ribosomes from E. culi, strain MRE 600, were prepared as described by Gesteland [4] , except that the cells were broken by glassbeads;.the standard buf- fer consisted of 10 mM MgAq, 5 mM tris (Cl), pH 7.4 and 6 mM mercaptoethanol. Prior to extraction of the protein, 70 S ribosomes were washed with 0.5 M NbCl solutions as described earlier [I] . Dissociation of 70 S ribosomes into 50 S and 30 S ribosomes was carried out by dialysis overnight against a solution of 0.1 mM MgAq, 5 mM tris (Cl), pH 7.6 and 6 mM mercaptoethanol. A 20 ml solution containing 300-500 mg ribosomal subparticles was layered on a linear sucrose gradient (lo-25% (w/v)) in the same buffer and centrifuged for 4 hr at 40,000 rpm at 3” in a Spinco B IV zonal rotor. The two types of subparticles were concentrated in a Diaflo apparatus, filter PM-30, and subsequently dialyzed overnight in the cold against 10 mM MgA%, 5 mM tris (Cl), pH 7.4,6 mM mercaptoethanol. The subparticles were pelleted in a Spinco Ti-50 rotor at SO,000 rpm for 5 hr and stored at -25”. Prior to extraction of the 50 S proteins, 50 S ribosomes were washed once with 0.5 M NH4CI solution [

Amino acid sequence of a 50 S ribosomal protein involved in both EFG and EFT dependent GTP‐hydrolysis

with a defined function are the two acidic proteins A, and A, (L, and L,,). Recently, much evidence has been accumulated that these two proteins of low molecular weight play an important role in the GTP-hydrolysis, associated with the elongation factors EFG and EFT [l-6]. Determination of the stoichiometry of ribosomal proteins indicates that 50 S ribosomes from logarith- mic growing cells contain on the average at least one copy each of A,- and AZ-protein per particle [7, 81. However, in conditions of restricted bacterial growth, under which ribosomes show an intrinsically reduced activity in protein biosynthesis [9, lo], the number of copies of A, -relative to A, -protein per ribosome decreases drastically [7, 111. In addition, particles reconstructed from “50 S cores”, supplemented with an excess of A, -protein, are significantly more active in promoting EFG- and EFT-factor mediated GTP- hydrolysis than similar particles constructed with the aid of A, -protein [ 1,6]. Since the only structural *

The primary structure of ribosomal protein eL12/eL12‐P from Artemia salina 80 S ribosomes

Protein eL12/eL12-P from Artemia salina 80 S ribosomes plays a role in the elongation step of protein synthetic reactions [l-3]. It is an acidic protein which was shown [2,3] to be partially phosphorylated on a single serine residue. Like the homologous proteins from procaryotic organisms, eL12/eL12-P from A. dina occurs in more than one copy per large subunit [3]; the isolated protein dimerizes in aqueous solutions [ 31. We reported on the amino-terminal part of this protein [4] and on a peculiar, ala&e-rich segment, located in the carboxy-terminal half of the protein chain [5]. Here, we wish to report the complete primary structure ofeL12/eL12-P from A. salina. This structure is compared with the published primary structure of protein HL20 from Halobacterium cutibtum [6]. Some possible, evolutionary relationships with its procaryotic L7/L12 counterpart are discussed.

Structure and phosphorylation of an acidic protein from 60S ribosomes and its involvement in elongation factor-2 dependent GTP hydrolysis

Summary 60S ribosomes from Artemia saline contain two acidic proteins eL12 and eL12(p) of closely related structure and function. The two proteins contain an identical amino acid sequence at the N-terminal region and they are immunologically indistinguishable. The N-terminal amino acid residue is in either case free methionine. Phosphorylation fully explains their slight difference in isoelectric point. No immunological relationship could be detected between eL12/eL12(p) and the 50S protein L7/L12 from Escherichia coli . Treatment of 80S ribosomes from Artemia saline with antibodies against eL12/eL12(p) results in inhibition of elongation factor-2 dependent GTP hydrolysis and polyphenylalanine synthesis. At least one other protein which is structurally related to eL12/eL12(p) is present in the large subunit of Artemia saline ribosomes. This additional protein is slightly larger, judged on the basis of SDS gel electrophoresis.

The human leucine zipper-containing guanine-nucleotide exchange protein elongation factor-1δ

Copy-DNA clones containing the complete coding region of the human elongation factor-1 delta (EF-1 delta) mRNA have been isolated and characterized. We present the deduced amino acid sequence and observe in it a leucine zipper motif seen recently in EF-1 delta from Artemia and Xenopus laevis. The human EF-1 delta sequence shows a strong conservation in its C-terminal domain. The homology between the N-terminal domains of EF-1 delta proteins is low and almost exclusively limited to the leucine zipper motif.

Sequence homology between EF-1α, the α-chain of elongation factor 1 from Artemia salina and elongation factor EF-TU from Escherichia coli

The occurrence, in the eukaryotic factor, of several ϵ‐trimethyllysine residues, is remarkable.