The yeast CLC protein counteracts vesicular acidification during iron starvation.
Ion gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to this family of intracellular exchangers. Gef1 localises to the late Golgi and prevacuole and is essential in conditions of iron limitation. In the absence of Gef1, a multicopper oxidase involved in iron uptake, Fet3, fails to acquire copper ion cofactors. The precise role of the exchanger in this physiological context is unknown. Here, we show that the Gef1-containing compartment is adjusted to a more alkaline pH under iron limitation. This depends on the antiport function of Gef1, because an uncoupled mutant of Gef1 (E230A) results in the acidification of the lumen and fails to support Fet3 maturation. Furthermore, we found that Gef1 antiport activity correlates with marked effects on cellular glutathione homeostasis, raising the possibility that the effect of Gef1 on Fet3 copper loading is related to the control of compartmental glutathione concentration or redox status. Mutational inactivation of a conserved ATP-binding site in the cytosolic cystathione beta-synthetase domain of Gef1 (D732A) suggests that Gef1 activity is regulated by energy metabolism.
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
Nikolai A
NA
Braun
Bruce
B
Morgan
Tobias P
TP
Dick
Blanche
B
Schwappach
D002570:Ceruloplasmin / Q000235:genetics / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D000090463:Iron Deficiencies; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018118:Chloride Channels; C089713:GEF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae; D005978:Glutathione