Occurrence of articulins and epiplasmins in protists

The cortex of ciliates. dinoflagellates, and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm in the flagellate Euglena gracilis and the ciliate Pseudomicrothorax dubius. The hallmark of articulins is a core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. The VPV-motif repeats are 12 residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. Using molecular and immunological tools we show that articulins are also present in the dinoflagellate Amphidinium carterae and the ciliates Paramecium tetraurelia and Paramecium caudatum, Tetrahymena pyriformis, and Euplotes aediculatus. Our analysis further shows that epiplasmins, a group of epiplasmic proteins first characterized in Paramecium, are also present in all these species. Moreover, we present evidence that epiplasmins and articulins represent two distinct classes of cytoskeletal proteins.     

The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins.

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.     

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p.

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.     

Plateins: a novel family of signal peptide-containing articulins in euplotid ciliates

In euplotid ciliates, the cortex is reinforced by alveolar plates--proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed alpha-, beta-, and gamma-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related beta- or gamma-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic alpha-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two alpha-platein genes from E. aediculatus. Even more acidic proteins, the derived alpha1- and alpha2-plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valine- and proline-rich 12-mers) are present in all three plateins. In beta/gamma-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the alpha-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of beta/gamma-platein, but near the N-terminus of alpha-plateins. All three plateins contain canonical N-terminal signal sequences, unique among known cytoskeletal proteins. The presence of start-transfer sequences correlates well with the final intra-alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin-related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre-fission morphogenesis are underway.     

Characterization of anther-expressed genes encoding a major class of extracellular oleosin-like proteins in the pollen coat of Brassicaceae†

A large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. íeven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3–5 mm B. napus buds, which contained microspores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32–38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N-terminal 15–20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of post-translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in the tapeturn of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed-specific intracellular oleosins, although their biological function may be different.     

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.     

The cDNA sequences of three tetrins, the structural proteins of the Tetrahymena oral filaments, show that they are novel cytoskeletal proteins

The oral filaments of the ciliate Tetrahymena consist of the tetrins, insoluble polypeptides with molecular masses of around 85 kD. We characterised the tetrins of T. thermophila by two-dimensional gels and derived a large number of peptide sequences by in gel digestion. Using RT-PCR techniques and RACE-PCR, the complete cDNA sequences of tetrins A, B and C were established. Although tetrins differ strikingly in protein sequence they show a common structural principle. A N-terminal domain of 60 to 100 residues contains most of the proline residues of the tetrins and is probably globular. It is followed by a long alpha-helical domain of 620 to 640 residues which either lacks prolines or in tetrin A contains a single proline residue. Although this long domain has coiled coil forming ability, the individual heptad repeats are not extensive. Tetrins are novel cytoskeletal proteins unique to ciliates. Since the three tetrin sequences account for all 900 amino acid residues obtained by microsequencing of peptides, an additional major tetrin seems excluded. A minor component D is related to tetrin B by peptide sequences. The isoelectric variants, particularly obvious for tetrin A, most likely reflect post-translational modifications. These could arise by phosphorylation of serines and threonines in the proline rich N-terminal domain.     

盐胁迫下苜蓿中盐蛋白的诱导产生

盐胁迫下苜蓿叶片中蛋白质的合成受到抑制,而其离体叶绿体中蛋白质合成增强,ABA阻碍了后者的蛋白质合成。NaCl胁迫下,“松江”和“肇东”两品种的根和叶中均无新多肽出现。在盐敏感的“松江”品种离体叶绿体中,NaGl诱导70,65,60和43kD4种多肽产生,ABA诱导60和17kD两种多肽产生;在较抗盐的“肇东”品种离体叶绿体中,NaGl诱导83,80kD和43kD3种多肽产生,但100mmol/L NaCl并不诱导83kD多肽出现,ABA无明显作用。两品种的43kD多肽和肇东品种的80kD多肽都存在于类囊体膜上,而松江品种的60kD多肽则存在于叶绿体间质中。     

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins.     

The human U5 snRNP-specific 100-kD protein is an RS domain-containing, putative RNA helicase with significant homology to the yeast splicing factor Prp28p.

Through UV-crosslinking experiments, we previously provided evidence suggesting that a U5 snRNP protein with a molecular weight in the 100-kDa range is an ATP-binding protein (Laggerbauer B, Lauber J, Lührmann R, 1996, Nucleic Acid Res 24:868-875). Separation of HeLa U5 snRNP proteins on 2D gels revealed multiple variants with apparent molecular masses of 100 kDa. Subsequent microsequencing of these variants led to the isolation of a cDNA encoding a protein with an N-terminal RS domain and a C-terminal domain that contains all of the conserved motifs characteristic of members of the DEAD-box family of RNA-stimulated ATPases and RNA helicases. Antibodies raised against cDNA-encoded 100-kDa protein specifically recognized native U5-100kD both on immunoblots and in purified HeLa U5 snRNPs or [U4/U6.U5] tri-snRNP complexes, confirming that the bona fide 100-kDa cDNA had been isolated. In vitro phosphorylation studies demonstrated that U5-100kD can serve as a substrate for both Clk/Sty and the U1 snRNP-associated kinase, and further suggested that the multiple U5-100kD variants observed on 2D gels represent differentially phosphorylated forms of the protein. A database homology search revealed a significant degree of homology (60% similarity, 37% identity) between the Saccharomyces cerevisiae splicing factor, Prp28p, which lacks an N-terminal RS domain, and the C-terminal domain of U5-100kD. Consistent with their designation as structural homologues, anti-Prp28 antibodies recognized specifically the human U5-100kD protein on immunoblots. Together with the DEXH-box U5-200kD protein (Lauber J et al., 1996, EMBO J 15:4001-4015), U5-100kD is the second example of a putative RNA helicase that is tightly associated with the U5 snRNP. Given the recent identification of the U5-116kD protein as a homologue of the ribosomal translocase EF-2 (Fabrizio P, Laggerbauer B, Lauber J, Lane WS, Lührmann R, 1997, EMBO J 16:4092-4106), at least three integral U5 snRNP proteins thus potentially facilitate conformational changes in the spliceosome during nuclear pre-mRNA splicing.     

Profilin tyrosine phosphorylation in poly-L-proline-binding regions inhibits binding to phosphoinositide 3-kinase in Phaseolus vulgaris

The profilin family consists of a group of ubiquitous highly conserved 12-15 kDa eukaryotic proteins that bind actin, phosphoinositides, poly-l-proline (PLP) and proteins with proline-rich motifs. Some proteins with proline-rich motifs form complexes that have been implicated in the dynamics of the actin cytoskeleton and processes such as vesicular trafficking. A major unanswered question in the field is how profilin achieves the required specificity to bind such an array of proteins. It is now becoming clear that profilin isoforms are subject to differential regulation and that they may play distinct roles within the cell. Considerable evidence suggests that these isoforms have different functional roles in the sorting of diverse proteins with proline-rich motifs. All profilins contain highly conserved aromatic residues involved in PLP binding which are presumably implicated in the interaction with proline-rich motif proteins. We have previously shown that profilin is phosphorylated on tyrosine residues. Here, we show that profilin can bind directly to Phaseolus vulgaris phosphoinositide 3-kinase (PI3K) type III. We demonstrate that a new region around Y72 of profilin, as well as the N- and C-terminal PLP-binding domain, recognizes and binds PLP and PI3K. In vitro binding assays indicate that PI3K type III forms a complex with profilin in a manner that depends on the tyrosine phosphorylation status within the proline-rich-binding domain in profilin. Profilin-PI3K type III interaction suggests that profilin may be involved in membrane trafficking and in linking the endocytic pathway with actin reorganization dynamics.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication

Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analysis of nuclear transport. Such cell- free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuclear pore. Here, the role of p97 has been investigated. Xenopus p97 was isolated and antisera were raised and affinity purified. Immunolocalization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior. Peptide sequence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is identical at 11/15 amino acids to a specific member of the GLFG family, NUP116. An additional peptide is highly homologous to a second sequence found in NUP116 and other members of the yeast GLFG family. A monoclonal antibody to the GLFG domain cross-reacts with a major Xenopus protein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family. The p97 antiserum was used to immunodeplete Xenopus egg cytosol and p97-deficient nuclei were reconstituted. The p97-depleted nuclei remained largely competent for nuclear protein import. However, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA. ssDNA replication in such extracts remains unaffected. Addition of the N-acetylglucosaminylated nuclear proteins of Xenopus or rat reverses these replication and growth defects. The possible role(s) of p97 in these nuclear functions is discussed.     

Identification and characterization of the major components of the Oncorhynchus mykiss egg chorion.

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.     

A novel human glycosyltransferase: primary structure and characterization of the gene and transcripts

We report the identification and primary structure of a novel human glycosyltransferase, B3GTL (beta3-glycosyltransferase-like). The 498 residue protein consists of a short cytoplasmic N-terminal "tail" (residues 1-4), a single transmembrane domain with type II topology (residues 5-28), a "stem" region (residues 29-260), and a catalytic domain (residues 261-498). The genomes of Anopheles gambiae, Drosophila melanogaster, and Caenorhabditis elegans encode potential orthologs which share 31-39% sequence identity with B3GTL, as well as the following features: a conserved catalytic domain containing a triple aspartate motif (DDD) at its core, a conserved pattern of cysteine residues, a C-terminal KDEL-like motif, and conserved residues and motifs that affiliate this novel group with a family of beta3-glycosyltransferases (GT31 in the CAZY classification). The B3GTL gene lacks canonical TATA and CAAT boxes and contains three functional polyadenylation sites. It is transcribed in a wide range of tissues and in TGF-beta-treated T84 epithelial cells.     

Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Characterization of Tiki,a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn²⁺/Co²⁺ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.     

The Human U5-220kD Protein (hPrp8) Forms a Stable RNA-Free Complex with Several U5-Specific Proteins, Including an RNA Unwindase, a Homologue of Ribosomal Elongation Factor EF-2, and a Novel WD-40 Protein

The human small nuclear ribonucleoprotein (snRNP) U5 is biochemically the most complex of the snRNP particles, containing not only the Sm core proteins but also 10 particle-specific proteins. Several of these proteins have sequence motifs which suggest that they participate in conformational changes of RNA and protein. Together, the specific proteins comprise 85% of the mass of the U5 snRNP particle. Therefore, protein-protein interactions should be highly important for both the architecture and the function of this particle. We investigated protein-protein interactions using both native and recombinant U5-specific proteins. Native U5 proteins were obtained by dissociation of U5 snRNP particles with the chaotropic salt sodium thiocyanate. A stable, RNA-free complex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unwindase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysis of the dissociated proteins. By cDNA cloning, we show that the 40kD protein is a novel WD-40 repeat protein and is thus likely to mediate regulated protein-protein interactions. Additional biochemical analyses demonstrated that the 220kD protein binds simultaneously to the 40- and the 116kD proteins and probably also to the 200kD protein. Since the 220kD protein is also known to contact both the pre-mRNA and the U5 snRNA, it is in a position to relay the functional state of the spliceosome to the other proteins in the complex and thus modulate their activity.     

Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.