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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Anion channels transport ATP into the Golgi lumen

Department of Cell and Developmental Biology, University of Colorado Health Sciences Center, Aurora, Colorado

Submitted 1 December 2004 ; accepted in final form 2 September 2005

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 K_m values 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^– > ATP^4– > MgATP^2–. Single-channel currents were observed at physiological concentrations of Cl^– and ATP, providing evidence for their importance in vivo. Second, transport of [-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 K_m = 25 µM. Finally, after transport of [-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, [-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.

Golgi complex; Cl channel; mass spectrometry; phosphorylation

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