Integral Protein Complex Research Articles

The RAD7, RAD16, and RAD23 genes of Saccharomyces cerevisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products.

Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions. In the yeast Saccharomyces cerevisiae, the RAD7, RAD16, and RAD23 genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcriptionally active genes. We have used a cell-free system to study the roles of the Rad7, Rad16, and Rad23 proteins in NER. Transcription-independent NER of a plasmid substrate was defective in rad7, rad16, and rad23 mutant extracts. Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex. Complementation studies with rad4, rad7, rad16, and rad23 mutant extracts suggest physical interactions among these proteins. This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16. Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro. Our results show that Rad7, Rad16, and Rad23 are required for transcription-independent NER in vitro. This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins.

The rabbit B cell antigen receptor is non-covalently associated with unique heteromeric protein complexes: Possible insights into the membrane IgM/IgD coexpression paradox

We describe several proteins that are components of the rabbit B cell receptor complex. Two proteins (37 kDa and 42 kDa) were found in non-covalent association with IgM expressed on B cells from peripheral blood. These proteins were also immunoprecipitated by anti-B29 (Ig-β) and anti-mb1 (Ig-α) monoclonal antibodies. As in the mouse and human, the IgM associated molecules were found as heteromeric structures with non-reduced apparent molecular weights of approximately 70–75 kDa. On rabbit B cells we also found these proteins in a 100–135 kDa complex which may represent trimeric or tetrameric structures. By Western blot, the 37 kDa protein was identified as rabbit Ig-β (B29), suggesting that the 42 kDa protein is rabbit Ig-α. These data suggest that rabbit IgM is associated with both Ig-α/β and Ig-(αβ) 2 or αβγ complexes. When similar immunoprecipitation studies were performed on lysates made from B cells isolated from appendix follicles, we found two additional IgM associated protein complexes containing 34 kDa and 36 kDa proteins.

Roles of proteasomes in cell growth

Proteasomes are large, unique protein complexes catalyzing energy- and ubiquitin-dependent proteolysis. Recent studies have revealed that these complexes are involved in two important cellular functions. One is to make antigen fragments for major histo-compatibility complex (MHC) class I-restricted antigen presentation and the other is to regulate the cell cycle by proteolysis. Here we review only the latter function of proteasomes. Proteasomes are widely distributed in eukaryotic cells, but their levels have been shown to be particularly high in various immature cells, such as cancerous, fetal and lymphoblastic cells, and agents including cell differentiation were found to suppress their expression. These conditions also regulate the expression of ubiquitin genes in a similar way, suggesting that proteasomes act ubiquitin-dependently in their 26S form in immature cells. High levels of proteasomes were found immunochemically in the nuclei of rapidly growing cells, indicating that proteasomes are important for eukaryotic cell growth. Indeed, gene disruptions of most subunits of proteasomes in yeast resulted in total suppression of cell growth and cell death. Short-lived regulatory factors of the cell cycle, such as Fos, p53, Mos, and cyclins are degraded by the proteasome-ubiquitin pathway under phosphorylated or dephosphorylated conditions. Ornithine decarboxylase, which is also a short-lived enzyme and is involved in the early phase of cell growth, is quickly degraded by proteasomes with antizyme, but without ubiquitination. Recently, we found that one of the regulatory factors of 26S proteasomes, p31, is a homologue of Nin1p, whose mutation caused inhibition of the cell cycle in yeast. These results indicate that proteasomes play important roles in regulation of the cell cycle in eukaryotes.

Distribution and genetic variabilities of mitochondrial plasmid-like DNAs in Paramecium.

Plasmid-like DNAs were isolated from mitochondria in many stocks of Para- mecium caudatum. Fifty-two among 68 stocks examined contained unique double-stranded DNAs (designated type I) ranging from 6.0 to 6.8 kb in length, and 12 stocks contained a set of 4 kinds of DNAs (8.2, 4.1, 2.8 and 1.4 kb; type II). Five of the stocks containing the type-I DNA had an additional 8.9-kb DNA (type III). No plasmid-like DNAs were found in the remaining 4 stocks. All these DNAs are linear and closely associated with proteins which can be removed only after proteinase-K digestion, suggesting that they exist as unique DNA protein complexes in mitochondria. Restriction and Southern blot analyses revealed a large sequence divergence among both type-I and -II DNAs. Type-I DNAs were found in every lineages of mitochondrial genomic DNA (mtDNA) which were inferred from restriction analyses. Type-II DNAs were, in contrast, scattered throughout the evolutionary tree of the mtDNAs. No obvious relations were found between the distribution of the DNAs and that of syngens (mating-type groups). Type-I DNAs were also found in other species of Paramecium, P. jenningsi and P. multimicronucleatum, and type-II DNAs in P. polycaryum.

Water cleavage by solar radiation?an inspiring challenge of photosynthesis research

Solar energy exploitation by photosynthetic water cleavage is of central relevance for the development and sustenance of all higher forms of living matter in the biosphere. The key steps of this process take place within an integral protein complex referred to as Photosystem II (PS II) which is anisotropically incorporated into the thylakoid membrane. This minireview concentrates on mechanistic questions related to i) the generation of strongly oxidizing equivalents (holes) at a special chlorophyll a complex (designated as P680) and ii) the cooperative reaction of four holes with two water molecules at a manganese containing unit WOC (water oxidizing complex) resulting in the release of molecular oxygen and four protons. The classical work of Pierre Joliot and Bessel Kok and their coworkers revealed that water oxidation occurs via a sequence of univalent oxidation steps including intermediary redox states Si (i = number of accumulated holes within the WOC). Based on our current stage of knowledge, an attempt is made a) to identify the nature of the redox states Si, b) to describe the structural arrangement of the (four) manganese centers and their presumed coordination and ligation within the protein matrix, and c) to propose a mechanism of photosynthetic water oxidation with special emphasis on the key step, i.e. oxygen-oxygen bond formation. It is assumed that there exists a dynamic equilibrium in S3 with one state attaining the nuclear geometry and electronic configuration of a complexed peroxide. This state is postulated to undergo direct oxidation to complexed dioxygen by univalent electron abstraction with YZ (ox) and simultaneous internal ligand to metal charge transfer.Key questions on the mechanism will be raised. The still fragmentary answers to these questions not only reflect our limited knowledge but also illustrate the challenges for future research.

Appearance and partial purification of a high molecular weight protein in crabs exposed to saxitoxin

K. G. Barber, D. D. Kitts, P. M. Townsley and D. S. Smith. Appearance and partial purification of a high molecular weight protein in crabs exposed to saxitoxin. Toxicon 26, 1027–1034, 1988.—This paper provides evidence for a protein component which appears to be involved in the seasonal resistance of small shore crabs, Hemigrapsus oregonesis and Hemigrapsus nudus to saxitoxin, a principle neurotoxin involved in paralytic shellfish poisoning (PSP). This unique protein complex was isolated and partially purified by ion exchange chromatography using DEAE - cellulose from visceral tissue extracts of resistant crabs. The complex was absent in control crabs that were sensitive to saxitoxin. In addition, the protein complex was induced in the crab after acute administration of low doses of saxitoxin. Results indicate that the protein complex is acidic in nature and has an apparent mol. wt of 145,000.

Characterization of electron transfer from water to plastocyanin catalyzed by resolved electron transfer complexes from chloroplasts

The photoreduction of soluble plastocyanin in a homologous reconstituted system requires a resolved Photosystem II preparation and the cytochrome b 6- f complex from spinach chloroplasts. Oxygen evolution is linked quantitatively to plastocyanin reduction in the reconstituted system, indicating that water is the electron donor for the photoreduction. The photoreduction is sensitive to inhibitors which specifically interact with the Photosystem II preparation [DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea] as well as those which interact with the cytochrome complex (DNP-INT: 2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether; and DBMIB: 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone). The plastocyanin requirement in the reconstituted system indicated a K m of 4.6 μ m and a V max of 4.2 μmol plastocyanin reduced per nmol cytochrome f per h. Inhibition curves for DNP-INT and DBMIB show that both are effective inhibitors (50% inhibition at a ratio of inhibitor to ctyochrome f of 1.0). The rate of reduction of plastocyanin was found to be stimulated by cations. This dependency was localized in the transfer of electrons from the Photosystem II preparation to the cytochrome complex. These results are considered in terms of organizational aspects of integral protein complexes in thylakoid membranes.

Reconstruction of the chloroplast noncyclic electron transport pathway from water to NADP with three integral protein complexes

RECONSTRUCTION OF PHOTOSYNTHETIC NONCYCLIC ELECTRON TRANSPORT FROM WATER TO NADP HAS BEEN ACCOMPLISHED BY USING THREE INTEGRAL PROTEIN COMPLEXES ISOLATED FROM CHLOROPLAST THYLAKOID MEMBRANES: photosystems I and II and the cytochrome b(6)-f complex. This system shows an absolute dependence on the presence of all three protein complexes for NADP reduction, in addition to plastocyanin, ferredoxin, and ferredoxin-NADP reductase. The reconstructed system was found to be sensitive to low concentrations of known inhibitors of noncyclic electron transport. Depletion of the Rieske iron-sulfur center and bound plastoquinone from the cytochrome b(6)-f complex resulted in an inhibition of the photoreduction of NADP.