Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex.

Import of the ADP/ATP carrier (AAC) into mitochondria requires the soluble TIM10 complex to cross the intermembrane space. We report here that Tim9 and Tim10 purified from Escherichia coli can form a complex of the same size as the endogenous complex from yeast mitochondria. This shows that no other mitochondrial protein is required for the formation of the TIM10 complex. Co-expression of both proteins rendered Tim9 more soluble and allowed purification of the reconstituted complex in a single step. Urea/EDTA treatment of recombinant Tim10 allowed its import into tim10-ts mitochondria that lack endogenous Tim10 and cannot import AAC. In this way, we were able to (i) reconstitute the TIM10 complex in the intermembrane space and (ii) restore import of AAC to almost wild-type levels. The reconstituted TIM10 complex not only facilitated passage of AAC across the outer membrane but also ensured its accurate membrane insertion. We conclude that the TIM10 complex can be formed exclusively from Tim9 and Tim10 and that the reconstituted complex efficiently restores AAC import in a strain lacking the TIM10 complex.

Origins of hydrogenosomes and mitochondria: evolution and organelle biogenesis.

It is becoming increasingly evident that all eukaryotes characterized to date bear some mitochondrial trait, whether it be a 'real' mitochondrion, a hydrogenosome, a mitosome or a few genes left behind from secondary losses of organelles. The implication is that the evolutionary history of the mitochondrion may reveal the history of the eukaryotic cell itself.

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria.

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.

Chromosomal organisation of a repeated gene cluster expressed in mammalian stages of Leishmania.

The genomic organisation of a large Leishmania gene cluster, expressed predominantly in intracellular, infective parasite stages, has been determined. Using cosmid cloning, parasite DNA fingerprinting, partial digestion and mapping with 'end-specific' probes, the Lm cDNA2 gene array has been localised to a 55-kb ClaI fragment within the L. major genome. Six tandemly linked gene copies are transcribed to produce an abundant 6-kb transcript; the seventh and last copy of the cluster is truncated at its 3' end. It is likely that these genes encode one or more proteins specific to infective stages of the parasite life cycle.