Isolation of Mitochondria from Ustilago maydis Protoplasts

Abstract

Ustilago maydis, a basidiomycete that infects Zea mays, is one of the top ten fungal models for studying DNA repair, signal transduction pathways, and dimorphic transitions, among other processes. From a metabolic point of view, U. maydis lacks fermentative capacity, pointing to mitochondria as a key player in central metabolism. Oxidative phosphorylation, synthesis of heme groups, Krebs cycle, β-oxidation of fatty acids, and synthesis of amino acids are some of the processes that take place in mitochondria. Given the importance of this organelle in eukaryotic cells in general, and in fungal cells in particular, we present a protocol for the isolation of U. maydis mitochondria based on the enzymatic disruption of U. maydis cell wall and differential centrifugation. The method can easily be extrapolated to other fungal species, by using appropriate lytic enzymes.

Keywords: Differential centrifugation; Lysing cell wall; Membrane potential; Mitochondria isolation; Respiratory chain; Ustilago maydis.

Figure 1.. Formation of Ustilago maydis protoplast.

During the incubation with lytic enzymes, the OD_600nm decreases due to the loss of the yeast cell wall. (A) In the first 30 min of incubation, 50% of the cells are converted to protoplast. (B) Because of the degradation of the cell wall, cells begin to form clumps.

Figure 2.. Oxygen consumption by U. maydis mitochondria.

Cells were grown in YPD liquid medium for 24 h at 28°C and collected by centrifugation. Then mitochondria were isolated as described in the protocol. Oxygen consumption was stimulated by adding A) succinate (10 mM), or B) NADH (0.15 mM), and the respiratory activity was inhibited by KCN (1 mM), or n-octyl gallate (2 µM).

Figure 3.. Spectral change of safranine caused by energization of mitochondria.

Mitochondria were energized by adding (A) 10 mM succinate (Succ), (B) 5 mM pyruvate-malate, or (C) 250 µM NADH. Then potential was abolished with 10 μM FCCP. The system contained: 300 mM sorbitol, 10 mM Hepes pH 7.0, 0.33 EGTA, 0.5 mg (protein) mitochondria, 0.2% BSA, and 2 μM safranine.