A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins

Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle. A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21. The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells. Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins. The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Secondary structure analyses of protein films on gold surfaces by circular dichroism

In order to analyze the secondary structures of protein molecules adsorbed on gold surfaces, circular dichroism (CD) spectra were measured and the secondary structure contents of protein ultra-thin films were estimated quantitatively. A disulfide group was introduced to cytochrome b(562) (cyt.b562), which is a water-soluble b-type heme protein. The cyt.b562 molecules self-assembled to form an ultra-thin protein film both on a gold substrate modified with 2,2(')-dithiodiacetic acid and on a bare gold surface. CD measurements were carried out both in solution and in air, and these results were compared. The protein denaturation was partially prevented, not only in solution but also in air, by both the modification of the substrate and the introduction of the anchor group to the protein molecule. The secondary structure contents of ultra-thin protein films on flat gold surfaces were observed for the first time both in solution and in air by CD spectra.     

A novel testis-specific RAG2-like protein,Peas: its expression in pachytene spermatocyte cytoplasm and meiotic chromatin

We report a novel gene Peas that constitutes an overlapping gene complex in mammalian genome. We have cloned human and mouse Peas cDNAs (hPEAS/mPeas) and analyzed their tissue and stage-specific expressions. Peas protein contains six repeated kelch motifs, structurally similar to RAG2, a V(D)J recombination activator, and is evolutionarily conserved among mammals, birds, insects, and nematodes. Northern, RNA in situ hybridization and immunohistochemical analyses showed that mPeas is specifically transcribed in testis, particularly in pachytene spermatocytes in which it is localized to the cytoplasm and meiotic chromatin. It is suggested that Peas may be involved in meiotic recombination process.     

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.     

Polymorphism in rice amylases at an early stage of seed germination

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In non-glutinous cultivars of rice, alpha-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha-amylases are repressed.     

Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.     

Regulation of the Gts1p level by the ubiquitination system to maintain metabolic oscillations in the continuous culture of yeast

Yeast cells exhibit sustained ultradian oscillations of energy metabolism in coupling with cell cycle and stress resistance oscillations in continuous culture. We have reported that the rhythmic expression of Gts1p is important for the maintenance of ultradian rhythms. Structurally, Gts1p contains sequence motifs similar to N-degron and the ubiquitin association domain, raising the possibility that the Gts1p level is regulated by degradation via ubiquitination. When the lysine residue at the putative ubiquitination site of the N-degron was substituted with arginine, both the protein level and half-life of mutant Gts1p increased. During continuous culture, the protein level of the mutant Gts1p was elevated and did not fluctuate, leading to the disappearance of metabolic oscillation within a day. Furthermore, using three Gts1ps containing mutations in the ubiquitin association domain, we showed that the lower the binding activity of the mutant Gts1ps for polyubiquitin in vitro, the higher the protein level in vivo. Expression of the mutant Gts1ps in the continuous culture resulted in an increase in Gts1p and early loss of the oscillation. Therefore, Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiquitinated Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillations.