The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Abstract

Mitochondrial F(1)F(O)-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F(1) sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0-9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5.

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

“…CI (rotenone-sensitive NADH:duroquinone oxidoreductase), CII+III (succinate:cytochrome c oxidoreductase) and CIV (cyanide-sensitive cytochrome c oxidase) activities were measured as described in Cardol et al (2002) and Remacle et al (2001). CV (oligomycin-sensitive ATP synthase) activity was measured as described in Villavicencio-Queijeiro et al (2009) based on the method from Pullman et al (1960). The specific activities were reported as the activity inhibited by maximal concentration of inhibitors used in vivo (see Results).…”

SDH6 and SDH7 Contribute to Anchoring Succinate Dehydrogenase to the Inner Mitochondrial Membrane in Arabidopsis thaliana

“…Furthermore, the mitochondrial processing peptidase is inserted into complex III of the mETC in plants (Braun et al, 1992). In the algal genus Polytomella, large additional subunits of mitochondrial ATP synthase form the scaffold for a stable dimerization of the enzyme complex (Villavicencio-Queijeiro et al, 2009).…”

Kinetic and hysteretic behavior of ATP hydrolysis of the highly stable dimeric ATP synthase of Polytomella sp.

“…dimeric ATP 222 synthase was 0.08 U/mg of protein. It has been previously reported223 that some detergents can activate the algal enzyme, particularly224 LM at a final concentration of 0.01%[17]. Saturation curves with225 Mg-ATP as substrate where performed for the dimeric ATP syn-226 thase and for the F 1 sector obtained after heat-treatment of the 227 intact enzyme[13], with or without detergent in the assay medium 228(Fig.…”

“…exhibits a very low 78 ATPase activity that increases upon heat treatment due to the 79 release of the F 1 sector. The hydrolytic activity of the dimeric 80 enzyme can also be unmasked by several non-ionic detergents 81 (lauryl maltoside, C 12 E 8 , Tween 20, Tween 80, and n-dodecanoyl 82 sucrose) [17]. Previous studies have shown that the fully functional 83 form of the enzyme is the dimer and that the monomeric assembly 84 represents a labile form with reduced ATPase activity and http://dx.doi.org/10.1016/j.abb.2015.03.018 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.…”

“…Previous studies have shown that the fully functional 83 form of the enzyme is the dimer and that the monomeric assembly 84 represents a labile form with reduced ATPase activity and http://dx.doi.org/10.1016/j.abb.2015.03.018 0003-9861/Ó 2015 Elsevier Inc. All rights reserved. 85 diminished oligomycin sensitivity [17]. In synthase were preincubated on ice for at least 30 min in the pres-139 ence of 10 lg/ml oligomycin (12.5 lM), unless stated otherwise.…”