Purification, primary structure, bacterial expression and subcellular distribution of an oocyte‐specific protein in Xenopus

Abstract

This study defines a novel Xenopus luevis protein (PIOO) that has recently been shown to be recognized by scleroderma patient sera. Using a combination of differential solubility in detergents, hydroxyapatite chromatography and one-dimensional PAGE, PlOO was purified to apparent homogeneity and the amino acid sequence was obtained. An oligonucleotide derived from this sequence was used to clone PlOO cDNA through a polymerase-chain-reaction cloning strategy. The entire PlOO cDNA sequence was determined, identifying a novel 83000-Da protein. Two alleles for PlOO were transcribed in the oocyte, with only one predicted amino acid change between them. Bacterial expression of a clone containing the entire PlOO coding region produced a protein that migrated at a mass 15% greater than that predicted from the amino acid sequence, indicating an aberrant electrophoretic mobility. The mRNA transcript for PlOO was only expressed during the previtellogenic stages of oogenesis (stages I and 11) and was absent from other Xenopus tissues. Similarly, the PlOO protein was found only in Xenopus oocytes and was localized to the cytoplasm of these cells. PlOO irreversibly bound single-stranded-DNA -cellulose but not double-stranded-DNA -cellulose. These data demonstrate the presence of a novel oocyte-specific protein in Xenopus.Autoantibodies in rheumatic diseases target a variety of highly conserved intracellular proteins, including those involved in RNA polymerase I and 111 transcription, heteronuclear RNA splicing, aminoacylation of certain tRNA species, DNA replication, DNA supercoiling and mitosis (review in [l]). The Xenopus oocyte and embryo have provided valuable systems for the characterization of many of these proteins. One example is the ribonucleoproteins targeted by systemic lupus erythematosus patient sera [2]. Antibodies against Sm and RNP were shown to inhibit intron excision from ribosomal genes when injected into germinal vesicles of oocytes, confirming that the RNA-splicing function of these particles demonstrated in vitro also occurred in vivo [3-51. Furthermore, Xenopus Sm and RNP were able to package exogenous small-nuclear RNA from mouse, Drosophila and yeast [6, 71. The Xenopus egg and early embryo were also shown to contain a large store of the U1 small nuclear RNA [6]. Fibrillarin, a ribonucleoprotein associated with U3 small nuclear RNA and recognized by scleroderma patient antibodies, was first cloned and sequenced from a Xenopus cDNA library [8, 91. Other autoantigenic proteins characterized in the Xenopus system include the proliferating cell nuclear antigen and the histones 130-131.Recent experiments in our laboratory have shown that scleroderma sera recognize a 100-kDa protein (P100) which is expressed in Xenopus luevis ovary [14]. Preliminary work also suggests a low level of expression in HeLa cells; however, attempts to clone a 100-kDa protein from a HeLa cell cDNA expression library with such sera have proven to be unsuccessful. In light of the abundance of a 100-kDa protein in X . luevis oocytes...

Distinct functions of maternal and somatic Pat1 protein paralogs

“…2, 3) for Western blotting of samples of oocytes, eggs, and embryos. On SDS-PAGE gels, xPat1a does not migrate according to its predicted size (83 kDa), but as a 100-kDa protein, as noted previously (Rother et al 1992), apparently due to aberrant SDS-binding (see Fig. 3B, below).…”

“…xPat1a is expressed throughout oogenesis but is not detectable in eggs, embryos ( Fig. 2A), nor in adult tissues (data not shown; Rother et al 1992). In contrast, xPat1b only starts to be expressed in late oogenesis (stage IV) and is relatively abundant in eggs and embryos, peaking at embryonic stages 12-20, corresponding to gastrula and neural fold stages ( Fig.…”

“…cerevisiae Pat1p (Pilkington and Parker 2008) and X. laevis P100 (Rother et al 1992), called xPat1a here, were used as queries in BLAST-P (NCBI). All the entries found were eukaryotic protein sequences.…”

“…The distantly related Xenopus homolog of yeast Pat1p (19% identity), called P100, was originally characterized as an oocyte-specific, cytoplasmic, ssDNA-binding protein (Rother et al 1992). More recently, Xenopus P100/Pat1 was identified by mass spectrometry as an abundant partner of CPEB and of Xp54 RNA helicase in oocytes, in coimmunoprecipitation and gel filtration analyses (Tanaka et al 2006;Minshall et al 2007).…”

Pat1 contributes to the RNA binding activity of the Lsm1-7–Pat1 complex

“…Like the Lsm1 through Lsm7 proteins, Pat1 is also well conserved in all eukaryotes. While a single Pat1 protein is present in yeast and invertebrates (Bonnerot et al 2000;Bouveret et al 2000;Tharun et al 2000;Eulalio et al 2007;Gallo et al 2008), vertebrates have two paralogs, Pat1a and Pat1b (Rother et al 1992;Scheller et al 2007;. Pat1 has been implicated in several functions, and not all of them are related to its association with the Lsm1-7 complex.…”