Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis.

We have cloned cDNAs encoding two variants of the elongation factor for protein synthesis in Xenopus laevis, called EF-1 alpha. One of these (42Sp50) is expressed exclusively in immature oocytes. It is one of two protein components of a 42S RNP particle that is very abundant in previtellogenic oocytes. The 42S RNP particle consists of various tRNAs, 5S RNA, 42Sp50 and a 5S RNA binding protein (42Sp43). A major function served by 42Sp50 appears to be the storage of tRNAs for later use in oogenesis and early embryogenesis. The second EF-1 alpha variant (EF-1 alpha O) is expressed mainly in oocytes but transiently in early embryogenesis as well. Its mRNA cannot be detected after neurulation in somatic cells. EF-1 alpha O is closely related to a third EF-1 alpha (EF-1 alpha S), discovered originally by Krieg et al. (1). EF-1 alpha S is expressed at low levels in oocytes but actively in somatic cells. The latter two proteins are very similar to known eukaryotic EF-1 alpha from other organisms and presumably function in their respective cell types to support protein synthesis.     

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.     

Stabilization of the maturation promoting factor (MPF) from Xenopus laevis oocytes. Protection against calcium ions

Maturation promoting factor (MPF) activity was recovered from progesterone-matured Xenopus oocytes cytosol, fractionated by polyethylene glycol-20,000 or ethanolic precipitation. An improved stabilization of the biological activity was obtained by adding adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) (50 microM) to the preparation buffers. Neither Ca2+ ions nor calmodulin inhibit the partially purified MPF.     

Elongation factor 1 beta of artemia: localization of functional sites and homology to elongation factor 1 delta

Elongation factor (EF)-1 beta, a 26 kDa protein, is the eukaryotic equivalent of bacterial EF-Ts, the nucleotide exchange factor in protein synthesis. EF-1 beta catalyzes the exchange of guanine nucleotides bound to EF-1 alpha; the latter protein is the eukaryotic equivalent of bacterial EF-Tu. Limited proteolytic cleavage studies on EF-1 beta 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-1 gamma, the third component of elongation factor 1. The regulatory, serine phosphate residue, Ser-89, localized in the hydrophilic stretch of EF-1 beta, 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-1 delta or 28 K protein, is homologous to EF-1 beta and contains regions very similar to the carboxyterminal part. EF-1 delta was found to be active in the nucleotide exchange reaction.     

Method for the preparation of active maturation promoting factor (MPF) from in vitro matured oocytes of Xenopus laevis.

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma

The β‐subunit of the human eukaryotic elongation factor 1 complex (heEF1β) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α‐ and γ‐subunits (eEF1γ). To biophysically characterize heEF1β, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL‐heEF1β), N‐terminus (NT‐heEF1β), and the C‐terminus (CT‐heEF1β) regions of the protein. Our results suggest that heEF1β is predominantly alpha‐helical and possesses an accessible hydrophobic cavity in the CT‐heEF1β. Both FL‐heEF1β and NT‐heEF1β form dimers of size 62 and 30 kDa, respectively, but the CT‐heEF1β is monomeric. FL‐heEF1β interacts with the N‐terminus glutathione transferase‐like domain of heEF1γ (NT‐heEF1γ) to form a 195‐kDa complex or a 230‐kDa complex in the presence of oxidized glutathione. On the other hand, NT‐heEF1β forms a 170‐kDa complex with NT‐heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL‐heEF1β associated with monomeric or dimeric NT‐heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (K_D = 9.6 nM for monomeric or 11.3 nM for dimeric NT‐heEF1γ). We postulate that the N‐terminus region of heEF1β may be responsible for its dimerization and the C‐terminus region of heEF1β modulates the formation of an ordered heEF1β‐γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.     

Affinity labeling of eukaryotic initiation factor 2 and elongation factor 1 alpha beta gamma with GTP analogs

As part of an attempt to understand the specific function and role of each subunit in multisubunit protein synthesis factors, we have attempted to identify the nucleotide binding peptides of eukaryotic initiation factor 2 (eIF-2). To ensure that the interactions were of a specific nature, two general controls were used: first, other protein factors with characterized GTP binding activity were tested; second, all affinity labeling was checked for nucleotide specificity by protection with the authentic nucleotide at a 10-fold molar excess over the affinity reagent. Results with a number of GTP modifying reagents ([alpha-32P]GTP, [alpha-32P]GDP, oxidized [alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GDP, and 5'-p-[8-3H]fluorosulfonylbenzoyl guanosine) indicate that appropriate conditions for both nucleotide and subunit specific labeling have been achieved. Under these conditions all reagents modified the beta subunit of eIF-2. Complementary studies with subunit-deficient forms of eIF-2 also suggest that the beta subunit of eIF-2 is involved with GTP binding. Coupled with other data suggesting that the gamma subunit of eIF-2 might be involved in GTP binding and amino acid sequence data of eIF-2 gamma from which a part of a GTP binding consensus sequence can be localized, support is provided for the concept of alternate GTP binding domains or a GTP binding domain shared between different subunits of eIF-2.     

A Gbetagamma stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.     

Mr 25 000 protein,a substrate for protein serine/threonine kinases,is identified as a part of Xenopus laevis vitellogenin B1

A phosphorylated protein with a molecular mass of 25 000 (pp25) previously purified from the cytosolic fraction of Xenopus laevis oocytes is an effective phosphate acceptor for casein kinases and protein kinase C. In this study, based on the partial amino acid sequence of pp25, a cDNA was isolated that encodes a new yolk precursor protein, Xenopus vitellogenin B1, which contained the sequence encoding pp25. Both mRNA and protein of vitellogenin B1 were expressed in all of the female organs examined. In agreement with a previous report, the amount of vitellogenin B1 protein in the liver increased after stimulation with estrogen. These results suggest that pp25 is a cytosolic non-crystallized yolk protein nutrient source, but it might also play a role in rapid development.     

Influence of cytochalasin B on oocyte maturation in Xenopus laevis

Cytochalasin B (CB) exerts an inhibiting effect on the formation, migration and anchoring in the cortex of the meiotic spindle in maturing Xenopus laevis oocytes. Regional sensitivity to CB (CB-sensitive zones) has been found in the oocytes which varies with reference to the stage of oocyte maturation at which CB is applied. Light and electron microscopy has shown that in these CB-sensitive zones the yolk and pigment granules, unlike the cortical ones, are displaced into the cytoplasm centripetally under the influence of CB.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

Purification and characterization of a germ cell-specific form of elongation factor 1 alpha (EF-1 alpha) from Xenopus laevis.

Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.     

An NPY-like peptide may function as MSH-release inhibiting factor in Xenopus laevis

This study demonstrates the presence of a rich plexus of neuropeptide Y (NPY) immunoreactive fibers in the hypothalamus and in the intermediate lobe of the pituitary of Xenopus laevis. During superfusion of neurointermediate lobe tissue, synthetic NPY induces a rapid, powerful and dose-dependent inhibition of in vitro release of MSH, endorphin and other proopiomelanocortin (POMC) derived peptides. Therefore, NPY undoubtedly is one of the growing number of neuropeptides that are likely involved in control of the amphibian MSH cells. Although a number of stimulatory neuropeptides have been found, this is the first neuropeptide to apparently function through an inhibitory mechanism. In that a 2-hr treatment with NPY did not influence POMC biosynthesis, nor processing of this prohormone to smaller peptides, we conclude that the primary action of NPY is a direct effect on the secretory process of the MSH cell.