Anion channels transport ATP into the Golgi lumen

Abstract

Anion channels provide a pathway for Cl−influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H+-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K2ATP added to the cis-chamber directly inhibited the Cl−subconductance levels of both channels, with Kmvalues ranging from 16 to 115 μM. Substitution of either K2ATP or MgATP for Cl−on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl−> ATP4−> MgATP2−. Single-channel currents were observed at physiological concentrations of Cl−and ATP, providing evidence for their importance in vivo. Second, transport of [α-32P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent Km= 25 μM. Finally, after transport of [γ-32P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [α-32P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl−and ATP influx into the Golgi lumen.