Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles

Abstract

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.

Fig. 1. Alignment of the novel Neocallimastix AAC protein reported here with typical members of the AAC subfamily, T.vaginalis hydrogenosomal HMP31, the maize brittle-1 protein and the human Graves’ disease carrier (GDC). Stars denote amino acids implicated in nucleotide binding or atractylate reactions in Bos taurus (Boulay et al., 1983; Dalbon et al., 1988); inverted triangles denote amino acids implicated in carrier function in S.cerevisiae (Gawaz et al., 1990; Lawson et al., 1990); shaded areas are signature motifs following each odd membrane spanning domain (Saraste and Walker, 1982); and boxed areas denote the six transmembrane helices.

Fig. 2. Phylogenetic analysis of AAC family protein sequences. Unrooted maximum likelihood phylogenetic tree of 32 AAC protein sequences. The Neocallimastix sequences are recovered as part of a monophyletic group otherwise defined by mitochondrial AACs of aerobic fungi. The polyphyly of green algal sequences is probably due to gene duplications early in the history of the AAC family, coupled with differential retention of paralogues among lineages (see text). Numbers at the nodes represent bootstrap values.

Fig. 3. Cellular fractionation and adenine nucleotide exchange in hydrogenosomal membranes. Western blot of N.patriciarum cellular fractions (20 µg) probed with an antiserum raised against N.crassa AAC (A) and the hydrogenosomal marker β-succinyl-CoA synthetase (Brondijk et al., 1996; Benchimol et al., 1997) (B). Lane 1: cell-free extract; lane 2: cytosolic fraction; and lane 3: hydrogenosomal fraction. (C) Uptake experiments with isolated hydrogenosomal membranes fused with liposomes. Fused membranes were loaded with 5 mM ADP and the exchange reaction was started by dilution of the vesicles in buffer containing a final concentration of 1.54 µM [8-¹⁴C] ATP (closed circles). Nucleotide exchange was inhibited by the addition of bongkrekic acid and carboxy-atractyloside at a final concentration of 20 and 25 µM, respectively (open circles).

Fig. 4. Expression of the hydrogenosomal ADP/ATP carrier of N.patriciarum in S.cerevisiae. (A) The S.cerevisiae strain WB-12 and transformants WB-12/pYES-Paac2-aacNpat (WB-12/AAC-Npat) and WB-12/pYES-Paac2-aac2Scer (WB-12/AAC2-Scer) were inoculated onto YP plates with 3% glycerol, and their growth was examined after 3 days at 30°C. (B) Western blot of isolated mitochondria (20 µg) from WB-12 (lane 1), WB-12/AAC-Npat (lane 2) and WB-12/AAC2-Scer (lane 3) probed with an antiserum raised against N.crassa AAC.

Fig. 5. ADP/ATP exchange in isolated mitochondrial membranes fused with liposomes. ADP/ATP exchange was measured by dilution of fused mitochondrial membranes loaded with 5 mM ADP into a buffer containing a final concentration of 1.54 µM [8-¹⁴C] ATP. (A) Uptake in mitochondrial membranes of S.cerevisiae strain WB-12 (open circles), WB-12/pYES-Paac2-aacNpat (closed circles) and WB-12/pYES-Paac2-aac2Scer (closed triangles). (B) The adenine nucleotide exchange activity in mitochondrial membranes of S.cerevisiae strain WB-12/pYES-Paac2-aacNpat in the presence of a 1000-fold excess of unlabelled substrate and expressed as a percentage of activity without added nucleotides. (C) Inhibition of adenine nucleotide exchange (closed circles) by the addition of 20 µM bongkrekic acid (closed triangles), 25 µM carboxy-atractyloside (open circles) and both (open triangles).