Purification and characterization of Mr 43,000 protein similar to Mr 25,000 protein, a substrate for protein Ser/Thr kinases, identified as a part of Xenopus laevis vitellogenin B1

Mr 25,000 protein (pp25), a substrate for protein Ser/Thr kinases, was recently shown to consist of a portion of the Xenopus laevis vitellogenin B1 protein. By Western blot analyses using antibodies against pp25, a minor protein band with Mr 43,000 (pp43) was detected in purified preparations of pp25. In this study, pp43 was highly purified through several column chromatography steps and its protein structure was analyzed. The amino acid sequence of the amino-terminal region of pp43 was the same as that of pp25. pp43 contained about two times more phosphates than pp25. These phosphates in pp43 were more resistant to acid phosphatase attack than those of pp25. pp43 was able to bind to pNiXa, a binding protein of pp25. Alpha-chymotryptic digestion generated a common fragment with molecular mass of 23,000 from both pp43 and pp25. These results suggest that pp43 may be a precursor of pp25 generated during processing of vitellogenin B1.     

Cellular distribution of Mr 25,000 protein, a protein partially overlapping phosvitin and lipovitellin 2 in vitellogenin B1, and yolk proteins in Xenopus laevis oocytes and embryos

A phosphorylated protein with molecular mass of 25,000 (pp25) can be derived from Xenopus laevis vitellogenin B1. In order to clarify the distribution of pp25, the changes in the concentration and localization of this protein in oocytes and embryos were examined by immunoblotting and immunohistochemistry using anti-pp25 antibodies, and compared with those of yolk proteins. In oocytes, pp25 was shown to localize characteristically at the surface just below the plasma membrane by immunohistochemical analysis. Interestingly, during embryogenesis, immunocytochemical staining revealed a transition of the pp25 distribution from beneath the outer surface of each germ layers to endoderm during tailbudding. In contrast, yolk proteins were localized in endoderm constantly throughout the developmental stages. However, the level of pp25 in the cytoplasm gradually decreased following the growth of embryos at the tailbud stage and disappeared at the tadpole stage, as shown by immunoblot analysis. These results suggest that pp25 could play different roles from those of yolk proteins such as lipovitellin and phosvitin in X. laevis oocytes and developing embryos.     

Purification and Characterization of M r 43,000 Protein Similar to M r 25,000 Protein, a Substrate for Protein Ser/Thr Kinases, Identified as a Part of Xenopus laevis Vitellogenin B1

M _r 25,000 protein (pp25), a substrate for protein Ser/Thr kinases, was recently shown to consist of a portion of the Xenopus laevis vitellogenin B1 protein. By Western blot analyses using antibodies against pp25, a minor protein band with M _r 43,000 (pp43) was detected in purified preparations of pp25. In this study, pp43 was highly purified through several column chromatography steps and its protein structure was analyzed. The amino acid sequence of the amino-terminal region of pp43 was the same as that of pp25. pp43 contained about two times more phosphates than pp25. These phosphates in pp43 were more resistant to acid phosphatase attack than those of pp25. pp43 was able to bind to pNiXa, a binding protein of pp25. α-chymotryptic digestion generated a common fragment with molecular mass of 23,000 from both pp43 and pp25. These results suggest that pp43 may be a precursor of pp25 generated during processing of vitellogenin B1.     

An estrogen-inducible protein binds specifically to a sequence in the 3'' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.     

Rudimentary phosvitin domain in a minor chicken vitellogenin gene

We have determined the nucleotide sequence and the derived amino acid sequence of the phosphoprotein-encoding region of the chicken vitellogenin III gene. The sequence of this minor vitellogenin could be aligned with exon 22 up to exon 27 of the previously sequenced major vitellogenin II gene (van het Schip et al., 1987). The exon 23 and 25 sequences are rich in serine codons (26% and 41%, respectively), and this region encodes at least one of the small egg yolk phosphoproteins. The major egg yolk phosphoprotein, phosvitin, is encoded by the analogous region in vitellogenin II. Comparison of the vitellogenin II and vitellogenin III sequences shows a great reduction in the size of the putative exon 23 of the latter (321 base pairs as opposed to 690). The number of serine codons is also drastically reduced from 124 in exon 23 of the vitellogenin II gene to 28 in vitellogenin III. The grouping of synonymous serine codons, as has hitherto been observed in sequenced vitellogenin phosphoproteins, has been maintained in vitellogenin III. A putative asparagine-linked N-glycosylation site which was conserved in the chicken vitellogenin II and the Xenopus laevis vitellogenin A2 gene, at the beginning of exon 23, is also present in vitellogenin III. The two chicken vitellogenins show a low conservation in the phosphoprotein-encoding region (average 33%, at the protein level) compared to that in the peripheral sequences (58% identity), which indicates that it is a rapidly evolving domain of the vertebrate vitellogenin gene.     

Mass-spectrometric Identification of Binding Proteins of <Emphasis Type="BoldItalic">M</Emphasis><Subscript>r</Subscript> 25,000 Protein,a Part of Vitellogenin B1, Detected in Particulate Fraction of <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.     

Placement of small lipovitellin subunits within the vitellogenin precursor in Xenopus laevis

N-terminal amino acid sequence data for the small lipovitellin subunits in Xenopus laevis crystalline yolk platelets indicate that LV2 beta is derived from vitellogenin A2 and that LV2 tau is most likely derived from vitellogenin A1. The small lipovitellin subunits apparently commence within the exon 24 region of the parental vitellogenins, flanking the C-terminal end of phosvitin. As a consequence, we conclude that most of the vitellogenin sequence encoded by exons 30 to 35 is not accounted for by the known yolk proteins.     

Apolipocrustacein,formerly vitellogenin,is the major egg yolk precursor protein in decapod crustaceans and is homologous to insect apolipophorin II/I and vertebrate apolipoprotein B

Background  

In animals, the biogenesis of some lipoprotein classes requires members of the ancient large lipid transfer protein (LLTP) superfamily, including the cytosolic large subunit of microsomal triglyceride transfer protein (MTP), vertebrate apolipoprotein B (apoB), vitellogenin (Vtg), and insect apolipophorin II/I precursor (apoLp-II/I). In most oviparous species, Vtg, a large glycolipoprotein, is the main egg yolk precursor protein.     

Modulation of Protein Phosphorylation by M r 25,000 Protein Partially Overlapping Phosvitin and Lipovitellin 2 in Xenopus laevis Vitellogenin B1 Protein

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. In an attempt to elucidate the physiological role of pp25, its effect on protein phosphorylation was studied. In vitro phosphorylation of some endogenous proteins from the cytoplasm and membrane fraction of Xenopus oocytes by casein kinase II and protein kinase C (PKC) was inhibited by increasing the concentration of pp25. By Western blot analysis using an antibody against phospho-(Ser/Thr) PKC substrate, phosphorylation of some endogenous proteins, especially in the cytoplasm, of Xenopus embryos was seen to increase when pp25 disappeared during developmental stages 35–45. These results suggest that pp25 may have a role as an inhibitory modulator of some protein phosphorylation in Xenopus oocytes and embryos.     

Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.     

Characterization of lipovitellin 2 as a tyrosine-phosphorylated protein in oocytes, eggs and early embryos of Xenopus laevis

A tyrosine-phosphorylated protein of 33 kDa is shown to be present in the solubilized yolk fraction of Xenopus laevis oocytes, eggs, and early embryos. The phosphoprotein is lipovitellin 2 as demonstrated by immunoprecipitation and mass spectrometry studies, and is termed pp33/LV2. Sub-cellular fractionation and immunoblotting studies demonstrate that pp33/LV2 is stably present in the Triton X-100-resistant and SDS-soluble yolk fractions during oogenesis, oocyte maturation, and early embryogenesis. From after the swimming tadpole stages (stage 39～), however, it becomes partly soluble to Triton X-100-containing buffer and all disappear thereafter (stage 48～49). In vitro enzyme assays with the use of the tyrosine phosphatase LAR and the tyrosine kinase Src demonstrate the reversible nature of the tyrosine phosphorylation of pp33/LV2. Microinjection studies demonstrate that the solubilized yolk fractions, but not those immunodepleted of pp33/LV2 or those pretreated with LAR, inhibit progesterone- or insulin-induced oocyte maturation. A pp33/LV2-like protein seems to present in two Xenopus subspecies, one other frog species, and two fish species, but not in other amphibian species, such as newt and salamander. These results suggest that LV2, in its tyrosine-phosphorylated form, serves in a cellular function in a species-specific manner, but the mechanism is still unknown.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.     

Degradation of M r 25,000 Protein by Cathepsin L-like Protease in Xenopus laevis Oocytes

A phosphorylated protein with a molecular mass of 25,000 (pp25) is involved in Xenopus laevis vitellogenin B1 and partially overlaps with phosvitin and lipovitellin 2. The protease responsible for pp25 degradation was studied in vitro since this occurs during embryogenesis. Initially, a protease thought to be a contaminant of the purified pp25 preparation was analyzed and an antipain-sensitive protease presumed to be involved. When commercially available proteases were examined, pp25 was not degraded by calpain I or 20S proteasome, but it was degraded by cathepsin L in vitro. A survey of the protease responsible for pp25 degradation in the cytoplasm of Xenopus oocytes found partially purified pp25 was degraded in partly antipain-sensitive manner. These results suggest that an antipain-sensitive protease or cathepsin L (or a related protease) is a candidate for pp25 degradation.     

Ferromagnetic isolation of endosomes involved in vitellogenin transfer into Xenopus oocytes

The transport pathway of the yolk precursor vitellogenin (VTG) has been followed using the techniques of ferrolabeling and ferromagnetic sorting, coupled with electron microscopic visualization. Vitellogenin conjugated to colloidal ferric particles of ca. 11 nm is selectively transported from the oolemma to the yolk platelets of vitellogenic Xenopus oocytes after gonadotropin stimulation of the female. Several cortical membrane compartments, labeled or unlabeled with ferric particles, are involved in the internalization and the transfer of vitellogenin to the yolk platelets. 1) Coated pits apparently fuse with coated vesicles, and coated vesicles fuse with each other in the outermost cortical cytoplasm. 2) Vesicles, depleted of their clathrin coat, fuse with cortical tubular endosomes and discharge their contents into yolk endosomes. 3) These endosomes are the direct precursors of the yolk organelles. 4) Endocytic vesicles fuse only with primordial yolk platelets of type I and not with type II or fully grown yolk platelets. After pulse-chase loading with ferric particles conjugated to vitellogenin and subsequent subcellular fractionation of the oocytes, ferromagnetic sorting of the various vesicle populations has been performed by using a "free-flow magnetic chamber". This novel method enables specification and characterization of purified endosomal compartments that accumulate protein yolk in Xenopus oocytes.     

VHDL, a larval storage protein from the corn earworm, Helicoverpa zea, is a member of the vitellogenin gene family

The hemolymph of last instar larvae of the corn earworm, Helicoverpa zea contains a blue very high-density lipoprotein (VHDL) that is selectively taken up into fat body prior to pupation. Its amino-terminal sequence was determined by Edman degradation, and used to design a degenerate primer for PCR amplification. With 5' and 3' RACE techniques, the entire cDNA coding for VHDL was amplified and sequenced. Conceptual translation reveals a 173 kDa protein that contains a 15 amino acid signal sequence immediately before the experimentally determined N-terminus of the mature protein. The protein contains a typical lipoprotein N-terminal domain, and shows high sequence similarity to vitellogenins from Lepidoptera and other insect species. VHDL mRNA was not detectable in adult H. zea, and antibodies raised against VHDL did not react with adult hemolymph or yolk proteins. Therefore VHDL, although a member of the vitellogenin gene family, seems to be distinct from the vitellogenin expressed in adult females.