RESUMO
A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.
Assuntos
Proteínas de Transporte/genética , Mitocôndrias/metabolismo , Proteínas de Protozoários/genética , Proteínas de Saccharomyces cerevisiae , Trichomonas vaginalis/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Metabolismo Energético , Evolução Molecular , Proteínas Fúngicas/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Dados de Sequência Molecular , Filogenia , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Trichomonas vaginalis/citologiaRESUMO
Hydrogenosomes are double membrane bounded redox organelles found in a number of amitochondriate protists and fungi. They are involved in carbohydrate metabolism and ATP synthesis and thus resemble mitochondria. Molecular analysis of the hydrogenosomal heat shock proteins Hsp70, Hsp60 and Hsp10 in Trichomonas vaginalis, one of the deepest-branching eukaryotes known to date, has revealed that these group exclusively with mitochondrial heat shock proteins. This finding indicates strongly that a progenitor organelle which gave rise to contemporary mitochondria and hydrogenosomes existed early in eukaryotic life. This hypothesis is further supported by similarities of hydrogenosomal and mitochondrial biogenesis. It was shown that T. vaginalis hydrogenosomal proteins are synthesized on free ribosomes in the cytosol with an N-terminal presequence that carries targeting information and is cleaved upon import into the organelle. Furthermore, as in mitochondrial import, hydrogenosomal protein import requires ATP, an electrochemical transmembrane potential and cytosolic protein factor(s). Here we demonstrate that inhibition of hydrogenosomal protein import occurs (i) in the presence of a synthetic presequence peptide and (ii) after pretreatment of hydrogenosomes with the protease trypsin. Trypsin pretreatment affects two hydrogenosomal membrane proteins of 31 and 70 kDa, respectively. Thus, we present evidence that import is saturable and that proteinaceous hydrogenosomal import receptor(s) exist. These results are a first step towards a characterization of the hydrogenosomal import machinery which should provide further insights into the relationship of hydrogenosomes and mitochondria and the evolution of protein targeting into organelles of endosymbiotic origin.
Assuntos
Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ferredoxinas/metabolismo , Genes de Protozoários , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Organelas/efeitos dos fármacos , Organelas/metabolismo , Precursores de Proteínas/metabolismo , Temperatura , Fatores de Tempo , Tripsina/farmacologiaRESUMO
Pair formation in the hypotrichous ciliate Euplotes octocarinatus is a poorly understood phenomenon. In order to obtain information about the molecules involved in this process, we compared ciliary and cell body-associated glycoconjugates of non-mating-competent, mating-competent, and conjugating cells. Detection of glycoconjugates was carried out on Western blots by immunostaining of oxidized, digoxigenin-labeled carbohydrate moieties. Using this method, in both of two complementary mating types a 130-kDa glycoprotein was identified, which appeared on cilia during acquisition of mating competence and was reduced during cell pairing. This suggests an active role of this glycoprotein in ciliary adhesion during pair formation. Additionally, in both of the two mating types a cell body-associated 135-kDa glycoprotein was detected, which is present in non-mating-competent cells as well as in mating-competent cells, but is strongly reduced in conjugating cells. In contrast to the ciliary 130-kDa glycoprotein, the cell body-associated 135-kDa glycoprotein is not surface-exposed [8]. We therefore propose that the cell body-associated glycoprotein is either involved in the preparation for cell fusion or meiosis or that it serves as a cytoplasmic pool for the ciliary 130-kDa glycoprotein.
Assuntos
Cílios/fisiologia , Conjugação Genética , Euplotes/fisiologia , Glicoproteínas/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Endopeptidase K , Peptídeos/fisiologia , Serina Endopeptidases/metabolismoRESUMO
Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont.
Assuntos
Proteínas de Protozoários/metabolismo , Trichomonas/metabolismo , Trifosfato de Adenosina/metabolismo , Alquilantes/farmacologia , Animais , Transporte Biológico , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Etilmaleimida/farmacologia , Ferredoxinas/metabolismo , Ionóforos/farmacologia , Leucina , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Temperatura , Trichomonas/efeitos dos fármacosRESUMO
Pheromone 3 mRNA of the ciliate Euplotes octocarinatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. No UAG codon was found. In connection with results reported for other ciliates, this suggests that the role of the classic termination codons had not yet been established when the ciliates started to diverge from other eukaryotes.