Anion channels transport ATP into the Golgi lumen

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 Km values ranging from 16 to 115 mu-M. 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 [{alpha}-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 mu-M. Finally, after transport of [{gamma}-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, [{alpha}-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. [PUBLICATION ABSTRACT]