Biochemical research on oogenesis: protein synthesis by purified 42S particles from Xenopus laevis and Tinca tinca previtellogenic oocytes

When incubated with ATP and a labeled amino acid, the 42S particles from early oocytes of Xenopus laevis and Tinca tinca incorporate radioactivity into tRNA and into a high molecular mass material which can be identified as protein. This incorporation is totally independent of ribosomes of cytosolic, mitochondrial or bacterial origin. The incorporated amino acids are linked to a broad spectrum of proteins by covalent bonds. Simple treatments such as incubation in buffer or addition of synthetic polyribonucleotides can inhibit the protein-labeling activity of the particles without affecting their tRNA aminoacylation activity. The former activity corresponds either to an amino acid polymerization reaction or to a protein-modifying reaction of a novel type. No involvement of mRNA in this process has been demonstrated. The alleged amino acid polymerization activity of the 42S particles could be a consequence of the conditions provided to aminoacyl tRNA by the tRNA-binding sites of the particles. These conditions are likely to allow the peptidyl transfer reaction to take place, although at a much lower rate than in the ribosome.     

Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes

Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopus oocytes is important for the regulation of germ layer formation and germ cell development. It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions of the mRNAs and with a number of different so-called localization proteins. In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection. Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1α (42Sp50). Incorporation of 42Sp50 into localization RNPs could be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50. Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs. Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm. Taken together, these observations provide evidence for a novel function of 42Sp50 in the context of vegetal mRNA transport in Xenopus oocytes.     

Two forms of elongation factor 1 alpha (EF-1 alpha O and 42Sp50), present in oocytes, but absent in somatic cells of Xenopus laevis

We have purified and partially sequenced the EF-1 alpha protein from Xenopus laevis oocytes (EF-1 alpha O). We show that the two cDNA clones isolated by Coppared et al. (Coppard, N. J., K. Poulsen, H. O. Madsen, J. Frydenberg, and B. F. C. Clark. 1991. J. Cell Biol. 112:237-243) do not encode 42Sp50, as claimed by these authors, but two very similar forms of EF-1 alpha O (EF-1 alpha O and EF-1 alpha O1). 42Sp50 is the major protein component of a 42S nucleoprotein particle that is very abundant in previtellogenic oocytes of X. laevis, 42Sp50 differs from EF-1 alpha O not only by its amino acid sequence, but also by several properties already reported. In particular, 42Sp50 has a low EF-1 alpha activity. It is distributed uniformly in the cytoplasm of previtellogenic oocytes, in contrast to EF-1 alpha O which is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body.     

Biochemical research on oogenesis. Binding of tRNA to the nucleoprotein particles of Xenopus laevis previtellogenic oocytes

In previtellogenic oocytes of Xenopus laevis, nearly all tRNA is included in nucleoprotein particles (thesaurisomes) sedimenting at 42 S. We evaluate the possibility of a tRNA exchange between the particles and the ribosomes during protein synthesis. We find that the particles take up tRNA after a very short incubation in vitro. In the absence of ATP, the particles preferentially bind charged tRNA. In the presence of ATP, more tRNA binds to the particles, and the sedimentation coefficient of the integrated tRNA is displaced to 45 S. When added to nonfractionated homogenates of oocytes together with ATP, poly(U) strongly stimulates the incorporation of radioactive phenylalanine into tRNA and protein. The labeled protein (polyphenylalanine) cosediments with the ribosomes, whereas most of phenylalanyl tRNA cosediments with the thesaurisomes. These data suggest that the thesaurisomes participate to some extent in protein synthesis. They release charged tRNA, thereby supplying the ribosomes with activated amino acids. Discharged tRNA is then taken up, reacylated, and stored in the particles until the next round of peptide bond formation. The aminoacylation and storage functions are probably carried out by two very unequal populations of particles. The main subclass of particles (42 S) binds and stores tRNA in an ATP-independent manner. A much smaller subclass of particles (45 S) is responsible for reacylation of discharged tRNA.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

Casein kinase II is a major protein phosphorylating activity in the nuclei of Xenopus laevis oocytes

The nuclei of Xenopus laevis oocytes contain kinases capable of phosphorylating endogenous and exogenous proteins using either ATP or GTP as phosphoryl donors. These enzymes are much more active with casein and phosvitin as substrates than with histones or protamines. The protein phosphorylating activity of oocyte nuclear extracts is not regulated by cyclic nucleotides, phorbol esters, calmodulin and calcium, or phospholipids. However, the casein phosphorylating activity can be greatly enhanced by the polyamines spermine or spermidine and drastically inhibited by heparin. Fractionation of the nuclear casein kinase activities by DEAE-Sephadex chromatography and glycerol gradient centrifugation indicate that the nuclei contain enzymes with the properties of casein kinases I and II as characterized in other species. Oocyte casein kinase I (Mr 37,000) is specific for ATP as phosphoryl donor, is only slightly inhibited by 10 micrograms/ml heparin, and is not significantly stimulated by polyamines. Casein kinase II (Mr 135,000) can use both ATP and GTP as substrates, and is very sensitive to heparin inhibition and polyamine stimulation. The fact that low concentrations of heparin (10 micrograms/ml) can inhibit a large percentage of the endogenous phosphorylation of nuclear extracts or of whole nuclei indicates that casein kinase II is probably the major protein phosphorylating activity of these oocyte organelles.     

Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.     

Biochemical research on oogenesis: distribution of tRNA-linked peptides and proteins in previtellogenic oocytes of Xenopus laevis

Peptides and proteins were detected in the deacylation products of tRNA purified from the 42S particles and from the messenger ribonucleoprotein particles (mRNPs) present in the previtellogenic oocytes of Xenopus laevis. Only a small fraction of particle tRNA carries a peptide or protein chain. The bulk of particle tRNA is simply aminoacylated. The tRNA-linked peptide chains of the particles appear to turn over more slowly in vivo than aminoacyl tRNA. These chains could arise in the particles by a peptidyl transfer reaction similar to that carried out by the ribosome.     

Symplekin,a constitutive protein of karyo- and cytoplasmic particles involved in mRNA biogenesis in Xenopus laevis oocytes

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo- and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV-VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the "Cajal bodies." However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3'-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Biochemical research on oogenesis. Comparison between the proteins of the ribosomes and the proteins of the 42 S particles from small oocytes of Xenopus laevis

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown.The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles.The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.     

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.     

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, IC, was activated by perfusing naive oocytes with a divalent-free recording solution. Ethanol (25-100 mM) modestly inhibited IC with 100 mM ethanol producing a 7-8% inhibition of steady state currents. The store-operated or capacitative calcium current (I(SOC)) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10-100 mM) dose-dependently inhibited the calcium-dependent chloride current (I(Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I(Cl(Ca)), I(Cl-1) and I(Cl-2). Under these conditions, ethanol (100 mM) inhibited I(Cl-1) currents to a greater extent (38%) than it did I(Cl-2) currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.     

Biochemical research on oogenesis. Transfer RNA is fully charged in the 42-S storage particles of Xenopus laevis oocytes.

1. Transfer RNA makes up 30-40% of total RNA in previtellogenic oocytes of Xenopus laevis. The bulk of tRNA is associated with 5-S RNA and two proteins in a high-molecular-weight complex sedimenting at 42S. 2. We show here that all kinds of tRNA are present in the 42-S particles and all of them sediment coincidently. Particle tRNA is fully charged in vivo. During purification of the 42-S particles tRNA becomes partially uncharged. When purified particles are incubated in vitro with amino acids and ATP a charging reaction occurs without disruption of the nucleoprotein complex. Many aminoacyl-tRNA synthetases can be shown to co-sediment with the 42-S particles. We conclude that complete aminoacylation of tRNA within the storage particles results from the activity of particle-bound aminoacyl-tRNA synthetases.     

42S p48--the most abundant protein in previtellogenic Xenopus oocytes--resembles elongation factor 1 alpha structurally and functionally.

We have undertaken an immunological and biochemical analysis of the most abundant soluble protein of previtellogenic Xenopus oocytes, 42S p48. We show that this protein shares immunological cross-reactivity with elongation factor 1 alpha (EF-1 alpha). Direct assays of both 42S fractions and purified 42S p48 show that this cross-reactivity is of functional significance since 42S p48, like EF-1 alpha, can transfer charged amino acids to ribosomes. We further demonstrate that 42S p48 is degraded soon after the onset of vitellogenesis, while the EF-1 alpha concentration remains essentially unchanged during this transition. These properties of 42S p48 are discussed with regard to its role in oogenesis.