Generation and purification of submitochondrial particles of different polarities from plant mitochondria

Abstract

Mitochondria were isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-salt medium. Submitochondrial particles (SMP) produced by sonication in a low salt medium + 20 mM MgCl 2 were 18% right-side-out (RO) as judged by the latency of cytochrome c oxidase assayed ± Triton X-100. SMP produced by French press treatment in a low-salt medium + 5 mM EDTA to remove bound divalent cations were 98% RO. Less extreme treatments gave SMP of intermediate polarity. There was a positive correlation between the % RO-SMP (produced by sonication) and the % NAD +-malate dehydrogenase enclosed within the SMP indicating that only RO-SMP contained trapped matrix. When a mixed population of SMP (45% RO) was applied to an aqueous polymer two-phase system, the top phase contained 76% RO-SMP and the bottom phase mostly inside-out SMP (26% RO). By analogy with the models for stacking of photosynthetic membranes, we propose that crista formation in the inner mitochondrial membrane is electrostatically regulated and SMP deriving from the closely stacked crista regions are inside-out.