A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by 31P nuclear magnetic resonance (NMR) spectroscopy. 31P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent Pi-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic Pi at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected Pi at a considerably lower pH of approximately 5.8-6.0. Pi at a considerably lower pH of approximately 5.8-6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two Pi-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the Pi-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to Pi in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated Pi was confirmed cytochemically.