Glucose and calcium‐responsive phosphatases mediate crosstalk between organelle and plasma membrane proton pumps

All eukaryotic cells require acidic organelles such as lysosomes, vacuoles, and endosomes maintained within a relatively alkaline cytosol for their proper function and survival. Disruption of pH homeostasis is observed in cancer, kidney diseases, and osteoporosis, as well as other diseases. Luminal pH of acidic organelles is primarily regulated by the highly conserved proton pump V‐ATPase, which also contributes to total cellular pH homeostasis by coordinating with cytosolic pH regulators at the plasma membrane. In yeast, pH homeostasis is maintained by the coordination of the organelle proton pump, V‐ATPase, and the essential, plasma membrane proton pump, Pma1. Interestingly, In yeast V‐ATPase loss of function ( vma ) mutants, ~ 50% of the Pma1 is endocytosed and degraded in the vacuole after ubiquitination by the E3‐ubiquitin ligase, Rsp5/Nedd4, aided by an α‐arrestin family adaptor, Rim8 ‐‐(Smardon & Kane, 2014). Failure to internalize Pma1 when V‐ATPase activity is compromised causes very poor growth, indicating that endocytic downregulation of Pma1 is compensatory. We seek to understand how loss of organelle acidification signals down‐regulation of Pma1. α‐arrestin family members, including Rim8, have been reported to be heavily phosphorylated, and several are activated by dephosphorylation. We find that two ser/thr phosphatases, calcineurin (PP2B), and Glc7 (PP1), are required for Pma1 endocytic down‐regulation in vma mutants. Calcineurin (CN) plays a central role in calcium homeostasis, and is activated in vma mutants because [Ca 2+ ] storage in the vacuole is compromised. A CN and V‐ATPase double mutant, cnb1Δvma2Δ, grows very slowly and fails to internalize Pma1. Treatment of vma2Δ cells with a CN inhibitor, FK506, prevents both Pma1 endocytosis and ubiquitination. This indicates that CN acts upstream of Pma1 ubiquitination to prevent Pma1 endocytosis. The essential phosphatase Glc7 bound to regulatory subunit Reg1 represses Pma1 activity under glucose starvation (Young et al. , 2010). The vma mutants have low cytosolic pH similar to glucose starved yeast cells, potentially activating Glc7‐Reg1 phosphatase activity. We find that a reg1Δ vma2Δ double mutant is inviable. A double glc7‐12 ts vma2Δ mutant is viable at permissive temperatures, but fails to grow at higher temperatures that are permissive for the individual mutations. At non‐permissive temperatures, the glc7‐12 ts vma2Δ double mutant also retains the Pma1 at the plasma membrane. We are currently working on determining the targets of CN and Glc7 in this pathway; potential targets are Pma1 itself and α‐arrestin adapter, Rim8. Taken together, our results suggest that vma mutants adapt to cellular pH imbalance by integrating signals from calcium and glucose homeostatic pathways to down‐regulate proton export via Pma1. Working model for ubiquitin‐mediated endocytic downregulation of Pma1 in yeast vma mutants: wt cells (left picture)vacuoles are acidic and cytosol is neutral. Under normal growth conditions, CN is inactive. Glc7 & reg1, potentially, not bound to each other. Importantly, Pma1 is at the PM. vma mutants (right picture) vacuolar pH rises & cytosolic pH decreases. Increased [Ca2+] cyto activates CN, & low cytosolic pH potentially activates reg1‐glc7 complex, both required for internalization of pma1.