Copper Blocks V-ATPase Activity and SNARE Complex Formation to Inhibit Yeast Vacuole Fusion

Abstract

The accumulation of Copper in organisms can lead to altered functions of various pathways, and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg + , Cd 2+ , and Cu 2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu 2+ to Cu + . Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl 2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H + pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu 2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects chelators. The Cu 2+ -specific chelator TETA rescued fusion, whereas the Cu + -specific chelator BCS had no effect on the inhibited fusion.

High throughput analysis of vacuolar acidification

“…Importantly, PMC family members as well as some TRP families can be inhibited by lanthanides [59,60,66]. In yeast Yvc1 releases Ca 2+ from the vacuole lumen during osmotic shock, however Yvc1 is unlikely involved here because it is not active under the conditions of the assay [15,[67][68][69]. If La 3+ and Gd 3+ inhibited Pmc1 (the primary Ca 2+ uptake transporter on the yeast vacuole), the extraluminal Ca 2+ would enter through the Ca 2+ /H + antiporter Vcx1 [70].…”

“…Vacuoles isolated from one liter of medium can yield upwards of 1 mg of vacuoles by protein. Importantly, these organelles contain all the essential proteins and lipids for in vitro analysis of fusion, fission, Ca 2+ transport, actin polymerization and V-ATPase activity [11,[14][15][16][17][18]. Furthermore, the relative ease and low cost of yeast genetics makes it easy to introduce gene deletions, mutations, truncations, and chimeras to further probe cellular functions including vacuole acidification [19,20].…”