The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis

The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica

Genetic Evidence for a Mitochondriate Ancestry in the ‘Amitochondriate’ Flagellate Trimastix pyriformis

Most modern eukaryotes diverged from a common ancestor that contained the α-proteobacterial endosymbiont that gave rise to mitochondria. The ‘amitochondriate’ anaerobic protist parasites that have been studied to date, such as Giardia and Trichomonas harbor mitochondrion-related organelles, such as mitosomes or hydrogenosomes. Yet there is one remaining group of mitochondrion-lacking flagellates known as the Preaxostyla that could represent a primitive ‘pre-mitochondrial’ lineage of eukaryotes. To test this hypothesis, we conducted an expressed sequence tag (EST) survey on the preaxostylid flagellate Trimastix pyriformis, a poorly-studied free-living anaerobe. Among the ESTs we detected 19 proteins that, in other eukaryotes, typically function in mitochondria, hydrogenosomes or mitosomes, 12 of which are found exclusively within these organelles. Interestingly, one of the proteins, aconitase, functions in the tricarboxylic acid cycle typical of aerobic mitochondria, whereas others, such as pyruvate:ferredoxin oxidoreductase and [FeFe] hydrogenase, are characteristic of anaerobic hydrogenosomes. Since Trimastix retains genetic evidence of a mitochondriate ancestry, we can now say definitively that all known living eukaryote lineages descend from a common ancestor that had mitochondria

Defense secretion of Prorhinotermes simplex: Toxicity to insecticide susceptible and resistant house fly

(E)-1-Nitropentadec-1-ene (NPE), the main component of the defense secretion of Prorhinotermes simplex soldiers, is toxic to both insecticide-susceptible (S) as well as to insecticide-resistant (R) strains of the house fly, Musca domestica. The LD50 is 11.7 mu g/female fly for the S strain and 9.7 mu g for the R strain. The same efficacy of NPE on R and S strains indicates a different mechanism of action compared to conventional chlorinated, organophosphorus, and pyrethroid insecticides. Termite nestmates are protected against NPE by a specific detoxification mechanism, 1-Nitropentadecane, the detoxification product of NPE is nearly nontoxic to house flies, and doses up to 160 mu g/fly caused only very low mortality

Identification, purification and separation of different isozymes of NADP-specific malic enzyme from Tritrichomonas foetus.

Tritrichomonas foetus was found to contain NADP-specific malic enzyme. The activity was present in the cytosolic fraction and was about 5-fold higher in extracts of a metronidazole-resistant strain (KV1-1MR-100) than of the parent strain (KVc1). Electrophoresis under non-denaturing conditions and activity staining indicated the existence of 3 isozymes termed I, II and III in order of increasing electrophoretic mobility. Isozymes I and II were much less active than isozyme III in the parent strain, whereas all three isozymes had comparable activities in the resistant strain. NADP-malic enzymes were purified from the cytosolic fraction of the resistant strain to apparent homogeneity and were identified by SDS-PAGE as polypeptides of 41.5 kDa (I), 40.5 kDa (III) and as a mixture of both in equal amounts (II). The molecular mass of the three holoenzymes was about 180 kDa, as determined by gel-filtration on Sephacryl S-300 HR, indicating a tetrameric structure. Isozyme III was also purified from parent strain and shown to consist of the 40.5-kDa polypeptide. Km values for malate were 0.31, 0.65 and 1.35 mM for isozyme I, II and III, respectively. From these results we conclude that T. foetus+, which is required for the formation of ethanol by alcohol dehydrogenase, an NADP-specific enzyme in this species. This is particularly important for the resistant strain, in which ethanol is the major end-product of glucose metabolism

Identification of a new lepidopteran sex pheromone in picogram quantities using an antennal biodetector: (8E,10Z)-tetradeca-8,10-dienal from Cameraria ohridella

A major sex attractant released by the virgin female of the horse-chestnut leafminer Cameraria ohridella Deschka et Dimic (Lepidoptera: Gracillariidae) which devastates horse-chestnut trees in Europe, was identified in picogram quantities as (8E,10Z)-tetradeca-8,10-dienal without using spectral methods. The identification solely relied on gas chromatography with electroantennographic detection (GC-EAD), calculation of Kovats' indices of the active principle on different GC phases, and construction of antennal response spectra (EAG response profiles) to C-12 and C-14 saturated and unsaturated standards with different functional groups. The dienal was prepared by a stereospecific synthesis and shown to be highly active for conspecific males in pg amounts and Fully comparable to the natural substance. (C) 1999 Elsevier Science Ltd. All rights reserved