Ubiquitin‐mediated endocytosis achieves pH homeostasis by facilitating communication between intracellular V‐ATPases and a plasma membrane proton exporter
Authors: Velivela Swetha Devi , Kane Patricia M 
Published: November 2023

Eukaryotic cells use ubiquitin‐mediated endocytosis for diverse functions such as attenuation of the GPCR signaling and downregulation of plasma membrane transporters. We study a signaling pathway involving ubiquitin‐mediated endocytosis in a new role, as a pH homeostatic mechanism. Disruption of pH balance is an underlying etiology of diseases such as cancer, osteoporosis, and metabolic acidosis. pH homeostasis at the cellular level is accomplished by different pH regulatory proteins, including V‐ATPases, proton pumps that acidify organelles such as vacuole/lysosomes, endosomes, and Golgi, and plasma membrane proton exporters. Together, these transporters keep cytosolic pH near neutrality. In S. cerevisiae, loss of V‐ATPase activity results in organelle alkalinization and signals downregulation of the essential, plasma membrane proton exporter, Pma1 through ubiquitin‐mediated endocytosis (Smardon and Kane (2015) JBC 290:27511). The E3‐ubiquitin ligase, Rsp5 and an α‐arrestin family adaptor protein, Rim8 are essential for the Pma1 internalization. When Pma1 internalization is prevented, mutants that lack V‐ATPase activity grow extremely poorly, indicating that Pma1 downregulation is growth compensatory. We hypothesize that Pma1 endocytosis rebalances overall pH homeostasis when V‐ATPase activity is compromised.In this study, we evaluated mechanisms for rebalancing pH homeostasis. First, we tested whether the compensatory effects of Pma1 downregulation require Pma1 endocytosis or can be achieved by simply lowering Pma1 activity at the plasma membrane. The pma1‐007 mutant has a promoter mutation that lowers Pma1 expression at the plasma membrane by 50%. We found that the pma1‐007 mutation is synthetically lethal in combination with a V‐ATPase mutation, vma2D. Because Pma1 is an essential gene, synthetic lethality could occur if Pma1 endocytosis still occurs in a pma1‐001 vma2Δ double mutant, resulting in plasma membrane Pma1 levels too low to support viability. Consistent with this, inhibition of V‐ATPase activity with concanamycin A triggers Pma1 endocytosis in pma1‐007 cells as well as wild‐type cells, indicating that vma mutants lack the ability to sense the level of Pma1 activity on the cell surface. Viability of a pma1‐007vma2D was not rescued by deleting RIM8, thus preventing Pma1 internalization; pma1‐007vma2D rim8D triple mutant is also inviable. Taken together, these results indicate that endocytosis of Pma1, and not simply the reduction of proton export activity, is required to rebalance cellular pH and allow viability in the absence of V‐ATPase activity. To better understand this phenomenon, we sought other candidates in the signaling pathway. Pma1 internalization requires a glucose responsive phosphatase, PP1, and a calcium‐responsive phosphatase, calcineurin. PP1 acts upstream of Rsp5‐Rim8 dependent ubiquitination of Pma1, whereas calcineurin may act further downstream, indicating a multi‐level regulation of the pathway. These signaling components have homologues in higher eukaryotes, so we propose that other eukaryotes may also use ubiquitin‐dependent endocytosis to ensure pH homeostasis.