RESUMO
The RNase P family is a diverse group of endonucleases responsible for the removal of 5' extensions from tRNA precursors. The diversity of enzyme forms finds its extremes in the eukaryal nucleus where RNA-based catalysis by complex ribonucleoproteins in some organisms contrasts with single-polypeptide enzymes in others. Such structural contrast suggests associated functional differences, and the complexity of the ribonucleoprotein was indeed proposed to broaden the enzyme's functionality beyond tRNA processing. To explore functional overlap and differences between most divergent forms of RNase P, we replaced the nuclear RNase P of Saccharomyces cerevisiae, a 10-subunit ribonucleoprotein, with Arabidopsis thaliana PRORP3, a single monomeric protein. Surprisingly, the RNase P-swapped yeast strains were viable, displayed essentially unimpaired growth under a wide variety of conditions, and, in a certain genetic background, their fitness even slightly exceeded that of the wild type. The molecular analysis of the RNase P-swapped strains showed a minor disturbance in tRNA metabolism, but did not point to any RNase P substrates or functions beyond that. Altogether, these results indicate the full functional exchangeability of the highly dissimilar enzymes. Our study thereby establishes the RNase P family, with its combination of structural diversity and functional uniformity, as an extreme case of convergent evolution. It moreover suggests that the apparently gratuitous complexity of some RNase P forms is the result of constructive neutral evolution rather than reflecting increased functional versatility.
Assuntos
Evolução Molecular , Deriva Genética , Precursores de RNA/genética , Ribonuclease P/genética , Arabidopsis , Catálise , Precursores de RNA/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiaeRESUMO
The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.
Assuntos
Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Escherichia coli/genética , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Ácido Oleico/farmacologia , Tamanho das Organelas , Peroxinas , Peroxissomos/ultraestrutura , Plasmídeos , Transporte Proteico , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , TransfecçãoRESUMO
Complete remission of BRAF V600E-driven ACC CUP by BRAF/MEK inhibition underscores importance of precision oncology.
Assuntos
Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Humanos , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Mutação , Masculino , Feminino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Analysis of selected cancer genes has become an important tool in precision oncology but cannot fully capture the molecular features and, most importantly, vulnerabilities of individual tumors. Observational and interventional studies have shown that decision-making based on comprehensive molecular characterization adds significant clinical value. However, the complexity and heterogeneity of the resulting data are major challenges for disciplines involved in interpretation and recommendations for individualized care, and limited information exists on how to approach multilayered tumor profiles in clinical routine. We report our experience with the practical use of data from whole-genome or exome and RNA sequencing and DNA methylation profiling within the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) program of the National Center for Tumor Diseases (NCT) Heidelberg and Dresden and the German Cancer Research Center (DKFZ). We cover all relevant steps of an end-to-end precision oncology workflow, from sample collection, molecular analysis, and variant prioritization to assigning treatment recommendations and discussion in the molecular tumor board. To provide insight into our approach to multidimensional tumor profiles and guidance on interpreting their biological impact and diagnostic and therapeutic implications, we present case studies from the NCT/DKFZ molecular tumor board that illustrate our daily practice. This manual is intended to be useful for physicians, biologists, and bioinformaticians involved in the clinical interpretation of genome-wide molecular information.
RESUMO
Dynamic changes of membrane structure are intrinsic to organelle morphogenesis and homeostasis. Ectopic expression of proteins of the PEX11 family from yeast, plant or human lead to the formation of juxtaposed elongated peroxisomes (JEPs),which is evocative of an evolutionary conserved function of these proteins in membrane tubulation. Microscopic examinations reveal that JEPs are composed of independent elongated peroxisomes with heterogeneous distribution of matrix proteins. We established the homo- and heterodimerization properties of the human PEX11 proteins and their interaction with the fission factor hFis1, which is known to recruit the GTPase DRP1 to the peroxisomal membrane. We show that excess of hFis1 but not of DRP1 is sufficient to fragment JEPs into normal round-shaped organelles, and illustrate the requirement of microtubules for JEP formation. Our results demonstrate that PEX11-induced JEPs represent intermediates in the process of peroxisome membrane proliferation and that hFis1 is the limiting factor for progression. Hence, we propose a model for a conserved role of PEX11 proteins in peroxisome maintenance through peroxisome polarization, membrane elongation and segregation.
Assuntos
Extensões da Superfície Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Peroxissomos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Extensões da Superfície Celular/patologia , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Engenharia Genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Peroxissomos/patologia , Ligação Proteica , Multimerização Proteica , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae , NicotianaRESUMO
Measuring Förster-resonance-energy-transfer (FRET) efficiency allows the investigation of protein-protein interactions (PPI), but extracting quantitative measures of affinity necessitates highly advanced technical equipment or isolated proteins. We demonstrate the validity of a recently suggested novel approach to quantitatively analyze FRET-based experiments in living mammalian cells using standard equipment using the interaction between different type-1 peroxisomal targeting signals (PTS1) and their soluble receptor peroxin 5 (PEX5) as a model system. Large data sets were obtained by flow cytometry coupled FRET measurements of cells expressing PTS1-tagged EGFP together with mCherry fused to the PTS1-binding domain of PEX5, and were subjected to a fitting algorithm extracting a quantitative measure of the interaction strength. This measure correlates with results obtained by in vitro techniques and a two-hybrid assay, but is unaffected by the distance between the fluorophores. Moreover, we introduce a live cell competition assay based on this approach, capable of depicting dose- and affinity-dependent modulation of the PPI. Using this system, we demonstrate the relevance of a sequence element next to the core tripeptide in PTS1 motifs for the interaction strength between PTS1 and PEX5, which is supported by a structure-based computational prediction of the binding energy indicating a direct involvement of this sequence in the interaction.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Sinais de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Competição entre as Células , Sobrevivência Celular , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Ligação Proteica , Domínios ProteicosRESUMO
Tetratricopeptide (TPR)-domain proteins are involved in various cellular processes. The TPR domain is known to be responsible for interaction with other proteins commonly recognizing sequence motifs at the C-termini. One such TPR-protein, TRIP8b, was originally identified in rat as an interaction partner of Rab8b, and its human orthologue as a protein related to the peroxisomal targeting signal 1 (PTS1) receptor Pex5p (Pex5Rp). Somewhat later, the mouse orthologue was reported to bind the hyperpolarization-activated, cyclic nucleotide-regulated HCN channels, and, very recently, the rat orthologue was shown to interact with latrophilin 1, the calcium-independent receptor of alpha-latrotoxin. Here we employed various methodological approaches to investigate and compare the binding specificities of the human PTS1 receptor Pex5p and the related protein Pex5Rp/TRIP8b towards a subset of targets, including Rab8b and various C-termini resembling PTS1. The results show that the TPR domains of Pex5p and Pex5Rp/TRIP8b have distinct but overlapping substrate specificities. This suggests that selectivity in the recognition of substrates by the TPR domains of Pex5p and Pex5Rp/TRIP8b is a matter of considerable complexity, and that no single determinant appears to be sufficient in unambiguously defining a binding target for either protein. This idea is further corroborated by our observations that changes in the surrounding residues or the conformational state of one of the binding partners can profoundly alter their binding activities. The implications of these findings for the possible peroxisome-related functions of Pex5Rp/TRIP8b are discussed.
Assuntos
Proteínas Oncogênicas/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Animais , Células CHO , Cricetinae , Cricetulus , Camundongos , Modelos Moleculares , Proteínas Oncogênicas/química , Receptor 1 de Sinal de Orientação para Peroxissomos , Ligação Proteica , Sinais Direcionadores de Proteínas , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Homologia Estrutural de Proteína , Proteínas rab de Ligação ao GTPRESUMO
HISTORY AND CLINICAL FINDINGS: A 72-year-old female presented with a therapy-resistant diarrhea. EXAMINATIONS: In the case of negative stool cultures and inconspicuous radiological imaging, further endoscopic diagnostics were performed. Histological implicated the image of a celiac disease in the duodenum and lymphocytic colitis reaching into the terminal ileum. In the case of negative antibody detection for celiac disease, a medication side effect was considered by differential diagnosis. TREATMENT: When olmesartan was discontinued, she developed a rapid improvement of the symptoms. CONCLUSION AND DIAGNOSIS: For the angiotensin receptor antagonist olmesartan, the occurrence of a sprue-like enteropathy has rarely described. Microscopic colitis is an exception.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/efeitos adversos , Diarreia , Imidazóis/efeitos adversos , Enteropatias , Tetrazóis/efeitos adversos , Idoso , Pressão Sanguínea , Doença Celíaca , Diagnóstico Diferencial , Diarreia/induzido quimicamente , Diarreia/complicações , Duodeno/efeitos dos fármacos , Duodeno/patologia , Feminino , Humanos , Enteropatias/induzido quimicamente , Enteropatias/complicaçõesRESUMO
Originally, the peroxisomal targeting signal 1 (PTS1) was defined as a tripeptide at the C-terminus of proteins prone to be imported into the peroxisomal matrix. The corresponding receptor PEX5 initiates the translocation of proteins by identifying potential substrates via their C-termini and trapping PTS1s through remodeling of its TPR domain. Thorough studies on the interaction between PEX5 and PTS1 as well as sequence-analytic tools revealed the influence of amino acid residues further upstream of the ultimate tripeptide. Altogether, PTS1s should be defined as dodecamer sequences at the C-terminal ends of proteins. These sequences accommodate physical contacts with both the surface and the binding cavity of PEX5 and ensure accessibility of the extreme C-terminus. Knowledge-based approaches in applied Bioinformatics provide reliable tools to accurately predict the peroxisomal location of proteins not yet determined experimentally.
Assuntos
Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Motivos de Aminoácidos , Animais , Humanos , Bases de Conhecimento , Oligopeptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Estrutura Terciária de Proteína , Especificidade da EspécieRESUMO
The glyoxylate cycle provides the means to convert C2-units to C4-precursors for biosynthesis, allowing growth on fatty acids and C2-compounds. The conventional view that the glyoxylate cycle is contained within peroxisomes in fungi and plants is no longer valid. Glyoxylate cycle enzymes are located both inside and outside the peroxisome. Thus, the operation of the glyoxylate cycle requires transport of several intermediates across the peroxisomal membrane. Glyoxylate cycle progression is also dependent upon mitochondrial metabolism. An understanding of the operation and regulation of the glyoxylate cycle, and its integration with cellular metabolism, will require further investigation of the participating metabolite transporters in the peroxisomal membrane.
Assuntos
Glioxilatos/metabolismo , Membranas Intracelulares/metabolismo , Peroxissomos/metabolismo , Sequência de Aminoácidos , Animais , Carbono/metabolismo , Compartimento Celular , Citosol/metabolismo , Proteínas Fúngicas/metabolismo , Redes e Vias Metabólicas , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes. The interaction between the PTS1 signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles. Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5-ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5. We found evidence that, at least the 12 C-terminal residues of a given substrate protein are implicated in PTS1 signal recognition. This motif can be structurally and functionally divided into three regions: (i) the C-terminal tripeptide, (ii) a region interacting with the surface of Pex5 (about four residues further upstream), and (iii) a polar, solvent-accessible and unstructured region with linker function (the remaining five residues). Specificity differences are confined to taxonomic subgroups (metazoa and fungi) and are connected with amino acid type preferences in region 1 and deviating hydrophobicity patterns in region 2.
Assuntos
Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Grupos de População Animal , Animais , Sítios de Ligação , Fungos , Humanos , Ligantes , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Solventes/química , Especificidade da EspécieRESUMO
Peroxisomal matrix proteins have to be imported into their target organelle post-translationally. The major translocation pathway depends on a C-terminal targeting signal, termed PTS1. Our previous analysis of sequence variability in the PTS1 motif revealed that, in addition to the known C-terminal tripeptide, at least nine residues directly upstream are important for signal recognition in the PTS1-Pex5 receptor complex. The refined PTS1 motif description was implemented in a prediction tool composed of taxon-specific functions (metazoa, fungi, remaining taxa), capable of recognising potential PTS1s in query sequences. The composite score function consists of classical profile terms and additional terms penalising deviations from the derived physical property pattern over sequence segments. The prediction algorithm has been validated with a self-consistency and three different cross-validation tests. Additionally, we tested the tool on a large set of non-peroxisomal negatives, on mutation data, and compared the prediction rate to the PTS1 component of the PSORT2 program. The sensitivity of our predictor in recognising documented PTS1 signal containing proteins is close to 90% for reliable prediction. The predictor distinguishes even SKL-appended non-peroxisomally targeted proteins such as a mouse dihydrofolate reductase-SKL construct. The corresponding rate of false positives is not worse than 0.8%; thus, the tool can be applied for large-scale unsupervised sequence database annotation. A scan of public protein databases uncovered a number of yet uncharacterised proteins for which the PTS1 signal might be critical for biological function. The predicted presence of a PTS1 signal implies peroxisomal localisation in the absence of N-terminal targeting sequences such as the mitochondrial import signal.
Assuntos
Algoritmos , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Análise de Sequência de Proteína , Motivos de Aminoácidos , Grupos de População Animal , Animais , Sítios de Ligação , Análise Mutacional de DNA , Bases de Dados de Proteínas , Fungos , Humanos , Camundongos , Modelos Moleculares , Receptor 1 de Sinal de Orientação para Peroxissomos , Proteínas/química , Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Reprodutibilidade dos Testes , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
Glyoxylate serves as intermediate in various metabolic pathways, although high concentrations of this metabolite are toxic to the cell. In many organisms glyoxylate is fed into the glyoxylate cycle. Enzymes participating in this metabolism are located on both sides of the peroxisomal membrane. The permeability of this membrane for small metabolites paves the way for exchange of intermediates between proteins catalyzing consecutive reactions. A model, in which soluble enzymes accumulate in close proximity to both ends of pore-like structures forming a transmembrane metabolon could explain the rapid and targeted exchange of intermediates. The metabolites passing the membrane differ between the three model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, and Candida albicans, which reflects the ease of evolutionary adaptation processes whenever specific transporter proteins are not involved. The atypical permeability properties of the peroxisomal membrane together with a flexible structural arrangement ensuring the swift and selective transport across the membrane might represent the molecular basis for the functional versatility of peroxisomes.
RESUMO
RNase P is the endonuclease that removes 5' extensions from tRNA precursors. In its best-known form, the enzyme is composed of a catalytic RNA and a protein moiety variable in number and mass. This ribonucleoprotein enzyme is widely considered ubiquitous and apparently reached its highest complexity in the eukaryal nucleus, where it is typically composed of at least ten subunits. Here, we show that in the protist Trypanosoma brucei, two proteins are the sole forms of RNase P. They localize to the nucleus and the mitochondrion, respectively, and have RNase P activity each on their own. The protein-RNase P is, moreover, capable of replacing nuclear RNase P in yeast cells. This shows that complex ribonucleoprotein structures and RNA catalysis are not necessarily required to support tRNA 5' end formation in eukaryal cells.
Assuntos
Núcleo Celular/enzimologia , Ribonuclease P/fisiologia , Trypanosoma brucei brucei/enzimologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genes de Protozoários/fisiologia , Genoma de Protozoário , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Complexos Multiproteicos/fisiologia , Organismos Geneticamente Modificados , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologia , Ribonuclease P/genética , Ribonuclease P/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Leveduras/enzimologia , Leveduras/genética , Leveduras/metabolismoRESUMO
The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.
Assuntos
Isocitrato Liase/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Ricinus communis/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Isocitrato Liase/genética , Dados de Sequência Molecular , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMO
BACKGROUND: Can sequence segments coding for subcellular targeting or for posttranslational modifications occur in proteins that are not substrates in either of these processes? Although considerable effort has been invested in achieving low false-positive prediction rates, even accurate sequence-analysis tools for the recognition of these motifs generate a small but noticeable number of protein hits that lack the appropriate biological context but cannot be rationalized as false positives. RESULTS: We show that the carboxyl termini of a set of definitely non-peroxisomal proteins with predicted peroxisomal targeting signals interact with the peroxisomal matrix protein receptor peroxin 5 (PEX5) in a yeast two-hybrid test. Moreover, we show that examples of these proteins - chicken lysozyme, human tyrosinase and the yeast mitochondrial ribosomal protein L2 (encoded by MRP7) - are imported into peroxisomes in vivo if their original sorting signals are disguised. We also show that even prokaryotic proteins can contain peroxisomal targeting sequences. CONCLUSIONS: Thus, functional localization signals can evolve in unrelated protein sequences as a result of neutral mutations, and subcellular targeting is hierarchically organized, with signal accessibility playing a decisive role. The occurrence of silent functional motifs in unrelated proteins is important for the development of sequence-based function prediction tools and the interpretation of their results. Silent functional signals have the potential to acquire importance in future evolutionary scenarios and in pathological conditions.
Assuntos
Motivos de Aminoácidos , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Sequência de Aminoácidos , Animais , Galinhas , Proteínas Fúngicas/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Muramidase/química , Muramidase/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Saccharomyces cerevisiae ANT1/YPR128c encodes the peroxisomal adenine nucleotide transporter that provides ATP for intra-peroxisomal activation of medium-chain fatty acids. A lacZ reporter construct comprising the ANT1 promoter was shown to be comparatively more highly expressed in a wild-type strain grown on oleic acid, a long-chain fatty acid, than in pip2Delta(oaf1)Delta mutant cells that are defective in fatty acid induction. The ANT1 promoter was demonstrated to contain a deviant oleate response element (ORE) that could bind the Pip2p-Oaf1p transcription factor and confer activation on a basal CYC1-lacZ reporter gene. Expression of Ant1p as well as other enzymes whose genes are known to be regulated by a canonical ORE was found to be increased in cells grown on lauric acid, a medium-chain fatty acid. We concluded that the signal for induction does not differentiate between long- and medium-chain fatty acids. This signal was independent of beta-oxidation or the biogenesis of the peroxisomal compartment where this process occurs, since a pox1Delta strain blocked in the first and rate-limiting step of beta-oxidation as well as various pex mutant cells devoid of intact peroxisomes produced sufficient amounts of Pip2p-Oaf1p for binding OREs in vitro and for expressing an ORE-driven reporter gene. The signal's durability was shown to be related to the concentration of fatty acids in the medium, since a pex6Delta strain expressed an ORE-driven reporter gene at high levels for a longer period than did isogenic wild-type cells. Generation of the signal was also independent of protein synthesis, as demonstrated by cycloheximide treatment.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ácidos Graxos/farmacologia , Genes Fúngicos , Proteínas de Transporte de Nucleotídeos , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Cicloeximida/farmacologia , Ácidos Graxos/química , Genes Fúngicos/efeitos dos fármacos , Genes Reporter , Óperon Lac , Ácido Oleico/farmacologia , Plasmídeos/genética , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.
Assuntos
Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Proteínas do Citoesqueleto/genética , Herança Extracromossômica , Meiose , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/fisiologia , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively. The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE. Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2. Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type. In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type. Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells. However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control. This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid. Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.
Assuntos
Ácido Oleico/metabolismo , Peroxissomos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/metabolismo , Northern Blotting , Divisão Celular , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Genes Reporter , Mutação , Oligonucleotídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de TempoRESUMO
The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid beta-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on different carbon sources. Immunoelectron microscopy in combination with cell fractionation showed that Mls1p was abundant in the peroxisomes of cells grown on oleic acid, whereas in ethanol-grown cells Mls1p was primarily cytosolic. This was reinforced using a green fluorescent protein (GFP)-Mls1p reporter, which entered peroxisomes solely in cells grown under oleic acid-medium conditions. Although growth of cells devoid of Mls1p on ethanol or acetate could be fully restored using a cytosolic Mls1p devoid of SKL, this construct could only partially alleviate the requirement for native Mls1p in cells grown on oleic acid. The combined results indicated that Mls1p remained in the cytosol of cells grown on ethanol, and that targeting of Mls1p to the peroxisomes was advantageous to cells grown on oleic acid as a sole carbon source.