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 K(2)ATP added to the cis-chamber directly inhibited the Cl(-) subconductance levels of both channels, with K(m) values ranging from 16 to 115 microM. Substitution of either K(2)ATP 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 [alpha-(32)P]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 microM. Finally, after transport of [gamma-(32)P]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-(32)P]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.
Thompson
Roger J
RJ
Dept. of Cell and Developmental Biology, Univ. of Colorado Health Sciences Center, Aurora, CO 80045, USA.
Akana
Hillary C S R
HC
Finnigan
Claire
C
Howell
Kathryn E
KE
Caldwell
John H
JH
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000873:Anthracenes / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000378:metabolism; D004594:Electrophysiology; D019286:Endopeptidase K / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D000873:Anthracenes; D018118:Chloride Channels; D002712:Chlorides; C508500:GOLAC-1 protein, rat; C508501:GOLAC-2 protein, rat; D007473:Ion Channels; C010030:9-anthroic acid; D000255:Adenosine Triphosphate; D019286:Endopeptidase K; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid