The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans
Fig 5
Impact of Vps45 on mitochondrial function.
A) and B) The association of Vps45-GFP with mitochondria was examined with the vps45 mutant complemented with VPS45-GFP. The strain was cultured in YNB and YNB + 150μM BPS for 24h at 30°C (A) and 37°C (B), cells were stained with 500nM MitoTracker Red CMXRos (mitotracker) and images were taken with a confocal microscope. C) The involvement of Vps45 in mitochondrial function and ROS sensitivity was examined by spot assays on inhibitors and H2O2. The WT, mutants and complemented strains were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing either 5mM salicylhydroxamic acid (SHAM), 3μg mL-1 antimycin A, 10mM potassium cyanide (KCN) or 0.01% H2O2. Plates were incubated at 30°C and 37°C for 2 days. D) Mitochondrial membrane potential was assessed by flow cytometry on 50,000 cells stained with JC-1 (5μM) and grown in iron deplete and replete conditions at 30°C (D) and 37°C (E). Experiments were carried out at least two times in triplicate (ANOVA ** P < 0.001).