Hidrogenosomas

Mycol. Res. 98 (2): 205-212 (1994)

Printed in Great Britain

205

Metabolic energy generation in hydrogenosomes of the anaerobic fungus Neocallimastix: evidence for a functional relationship with mitochondria

FEMKE D. MARVIN-SIKKEMA"", ARNOLD J. M. DRIESSEN, JAN C. GOTTSCHAL AND RUDOLF A. PRINS
Department of Microbiology. University of Groningen. Kerklaan 30. 9751 NN Haren. TheNetherlands

Anaerobic eukaryotes are often devoid of mitochondria but contain special organelles separated from the cytosol by a single (in fungi) or a double (in protozoa) membrane. Hydrogenosomes from the anaerobic fungus Neocallimastix sp. L2 are thought to catalyse the enzymic steps in the ATP-yielding metabolism of malate into acetate. H 2 and CO 2 , Isolated hydrogenosomes contain a Mg 2 +- orMn 2 +-dependent ATPase activity. This activity is involved in the maintenance of a pH gradient across the hydrogenosomal membrane. which renders these organelles alkaline inside. ATPase activity and .b.pH generation is sensitive to diethylstilboestrol but not to other known ATPase inhibitors. Typical inhibitors of the mitochondrial ADP I ATP translocase. bongkrekic acid and carboxyatractylatereduced the ATPase activity. suggesting the presence of a nucleotide transporter. Under anaerobic conditions hydrogenosomes produced H 2 and acetate from malate. This process was found to be dependent on the external supply of ATP or ADP and succinate, and was blocked by protonophores. diethylstilboestro\, and the inhibitors bongkrekic acid and carboxyatractylate. These results demonstrate thathydrogenosomes of Neocallimastix sp. L2 perform the essential functions required for the generation of metabolic energy from malate. It is suggested that hydrogenosomes are functionally related to mitochondria but lack an outer membrane.

Anaerobic fungi are important inhabitants of the digestive tract of many herbivorous mammals (Orpin & Joblin, 1988; Milne et ai., 1989). They playa role in thedegradation of plant material by the production of cell wall-degrading enzymes (Williams & Orpin, 1987) and by physically weakening the cell wall structure with their rhizoidal system (Akin et ai., 1989). Anaerobic fungi and many other anaerobic eukaryotes, such as trichomonad protozoa and (rumen) ciliates, contain special organelles, termed 'hydrogenosomes'. A major share of the metabolism leading tothe production of H 2 , CO 2 and acetate is thought to be localized in these organelles (MarvinSikkema et ai., 1993b; Miiller, 1988; YarleH et ai, 1986). Characterization of the metabolic functions of these organelles is impaired by the liability to molecular oxygen and the difficulty of obtaining stable and intact hydrogenosomes. The recent development of a rapid procedure for the isolation ofintact hydrogenosomes from the anaerobic fungus Neocallimastix sp. L2 (Marvin-Sikkema et ai.. 1993 b) enables us for the first time to study the function of these organelles in detail. When grown on glucose as sole energy source, Neocallimastix sp. L2 produces H 2 and CO 2 , These products are presumed to be formed from malate. which is thought to be a substrate for hydrogenosomes (Marvin-Sikkema etai., 1993 b). Hydrogenosomal metabolism also leads to the
• Corresponding author. Present address: Division of Industrial Microbiology. Department of Food Science. Wageningen Agricultural University. P.O. Box 8129. 6700 EV Wageningen. The Netherlands.

production of ATP at substrate level. The energy-generating function of the hydrogenosomes is further emphasized by their localization in thevicinity of the flagellar pole in the zoospores of Neocallimastix sp. L2 (Marvin-Sikkema et ai., 1992, 1993 b). Hydrogenosomes may present the energy to the flagellar motors required for locomotion. Mitochondria are absent in this organism (Marvin-Sikkema et ai.• 1992), and are possibly replaced by the hydrogenosomes, suggesting a functional relationship between these organelles. On the other...