Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.
Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).
Department of Biophysics, Wageningen University, The Netherlands. stephan.hesse@algeeman.mgim.wau.nl
Stephan J A
SJ
Hesse
George J G
GJ
Ruijter
Cor
C
Dijkema
Jaap
J
Visser
D000963:Antimetabolites / Q000494:pharmacology; D001234:Aspergillus niger / Q000737:chemistry / Q000502:physiology*; D050260:Carbohydrate Metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D019343:Citric Acid / Q000378:metabolism; D003199:Computer Systems; D004734:Energy Metabolism; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D007476:Ionophores / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D019810:Sodium Azide / Q000494:pharmacology; D014617:Vacuoles / Q000737:chemistry
D000963:Antimetabolites; D007476:Ionophores; D019343:Citric Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D019810:Sodium Azide