Mitochondria from conidia of Neurospora crassa were isolated and purified by a simple and rapid technique. Electron micrographic analysis and isopycnic centrifugation on a sucrose density gradient revealed a heterogeneous pbpulation.Succinate, citrate, 2-oxoglutarate, NADH and ascorbate + tetramethyl-pphenylenediamine (TMPD) were oxidized by the mitochondria. Oxygen consumption was totally inhibited by antimycin A or cyanide. The electron transport chain was coupled to sites 1 and 2 of phosphorylation. Oligomycin, atractyloside and 2,4-dinitrophenol prevented energy coupling. I N T R O D U C T I O N Respiratory properties of mitochondria isolated from mycelia at different growth stages of Neurospora crassa wild type are now well characterized (Hall '8z Greenawalt, 1967; Weiss et al., 1970; LamboWitz et al., 1972a, b; von Jagow et al., 1973).It has been reported that conidial mitochondria of N. crassa have a low rate of oxygen consumption and that this respiration is predominantly cyanide insensitive (Colvin et al., 1973) and fails to perform oxidative phosphorylation (Greenawalt et al., 1972). However, in a preliminary study, Hall 8z Greenawalt (1964) showed that mitochondria extracted from conidia of N. crassa were able to carry out oxidative phosphorylation. To clarify this discrepancy, we have developed an improved procedure for the extraction of conidial mitochondria of N. crassa (Ortega Perez et al., 1977) which enables us to isolate large quantities of relatively pure mitochondria which show structural integrity -and well-preserved respiratory functions. After extraction under these improved-conditions, conidial mitochondria possess a functional electron transport chain which is partially coupled to oxidative phosphorylation and totally sensitive to cyanide and antimycin A. Heterogeneity of mitochondria was observed by electron microscopy and isopycnic centrifugation.
M E T H O D SOrganism and cultural conditions. The wild type of Neurospora crassa strain STA, 262A was obtained from the Fungal Genetic Stock Center, Humboldt State University, Arcata, California, U S A . Conidia were produced by inoculating 1.8 1 Fernbach flasks containing 200 ml Neurospora Minimal Medium (Difco) and 2% (w/v) agar with 2 x lo* conidia. Germination and growth proceeded for 2 d in the dark at 25 OC; the cultures were then kept for 3 d at the same temperature under continuous fluorescent light and aerated with humidified air.Cell disruption and mitochondria1 preparation. Conidia of N. crassa were harvested with distilled water, filtered through four layers of gauze and concentrated by centrifugation at 2 500 g for 10 min. The conidial pellet was further washed with distilled water and reconcentrated by low-sped centrifugation. Conidia were disrupted on a Vortex mixer modified as follows: the rubber ring was replaced by a disc of PVC (3 mm thick) with eight 0022-1287/81/0000-9864 $02.00 0 1981 SGM
