How to Isolate Tightly-Coupled Mitochondria from Neurospora crassa mycelium. A Fast and Reproducible Procedure

Substation genetic and molecular information has been obtained after 70 years of intense research with Neurospora which provided foundations for studying mitochondrial activities and biogenesis.[Davies, 2002; Borkovich et al., 2004]. The basic understanding of mitochondrial operation has been enhanced tremendously in recent years by advances in structural biology that span the resolution spectrum. X-ray crystallography of large soluble and integral membrane protein complexes is providing atomic-level detail of the mitochondrial respiratory machinery, promising to unravel the mechanisms of electron transfer, proton pumping, and phosphorylation. However, a frustrating limitation is the lack of a technique allowing for the isolation of fairly intact mitochondria at different growth stages [see Sebald et al., 1979; Lambowitz et al., 1972a; Lambowitz and Slayman, 1972; Schwitzguebel and Palmer, 1982; Cramer et al., 1983; Bowman and Bowman, 1988]. Therefore, methods for the rapid and large-scale isolation of physiologically intact organelles from Neurospora mycelium ought to be of general value. Here, we report an improved procedure for preparation of fairly intact mitochondria from N. crassa mycelium at different growth phases. We empirically optimized the composition and concentrations of the lytic enzymes, the time required for spheroplast formation, and the composition of washing and grinding media. In addition, the whole procedure was significantly simplified and shortened to 2.5 h. Thus, the distinctive advantages of the protocol proposed are its simplicity, reproducibility, and versatility. The mitochondria preparations isolated from mycelium harvested from exponential and stationary growth phases respired actively with Krebs cycle intermediates, exogenous NADH, and some other substrates. They met all known criteria of physiological intactness; they controlled metabolic states upon successive additions of ADP, displayed good respiratory control (reflecting the degree of oxidation to phosphorylation coupling) and were close to the theoretically expected ADP/O values (reflecting the efficiency of the oxidative phosphorylation) (see Table 1). RC and ADP/O ratios upon oxidation of all examined substrates were higher than previously reported [Lambowitz et al., 1972b]; moreover, RC ratios, obtained upon oxidation of 2-oxoglutarate attained record values, far exceeding those reported not only for N. crassa mitochondria, but also for mitochondria from other fungi. Ficoll-purified mitochondria displayed 2-fold higher respiratory rates, retaining almost the same RC and ADP ratios (not shown). Mitochondria contained the fully competent respiratory chain with all three points of energy conservation. Oxidation of all examined substrates by mitochondria from WT was mediated by two alternative terminal oxidative