# Oxr1 and Ncoa7 regulate V-ATPase to achieve optimal pH for glycosylation within the Golgi apparatus and trans-Golgi network

## TL;DR

The proteins Oxr1 and Ncoa7 regulate Golgi/TGN pH by inhibiting V-ATPase, ensuring optimal conditions for glycosylation and organelle function.

## Contribution

Oxr1 and Ncoa7 are identified as Rab-binding proteins that inhibit V-ATPase to regulate Golgi/TGN pH.

## Key findings

- Oxr1 and Ncoa7 localize to the Golgi/TGN and inhibit V-ATPase activity via their TLDc domains.
- Depletion of Oxr1 and Ncoa7 causes Golgi/TGN acidification and impaired protein glycosylation.
- Oxr1 and Ncoa7 depletion also affects lysosomal pH and membrane integrity.

## Abstract

In the secretory pathway, the pH within organelles is gradually acidified, starting at 7.1 to 7.2 in the endoplasmic reticulum, decreasing to 6.0 to 6.7 in the Golgi apparatus/trans-Golgi network (Golgi/TGN), and reaching 4.5 to 5.0 in lysosomes. Acidic pH is maintained by the vacuolar-type ATPase (V-ATPase) present at the Golgi, lysosomes, and other organelles in the late secretory and endocytic pathways; however, the mechanism underlying this pH variation within different organelles has not been elucidated. Here, we identified the TLDc domain-containing proteins Oxr1 and Ncoa7 as Rab-binding proteins that localize to the Golgi/TGN membrane and regulate luminal pH by inhibiting V-ATPase activity. Our findings explain why the pH in the Golgi/TGN is less acidic than that in lysosomes.

Maintenance of pH within membranous organelles is crucial for cellular processes such as posttranslational modifications, ligand–receptor interactions, and proteostasis. The precise mechanisms that determine the luminal pH of each organelle are not fully understood. This study investigated the mechanisms that regulate luminal pH to ensure optimal enzymatic activity. We identified Oxr1 and its paralog Ncoa7, which regulate the vacuolar-type proton pump ATPase (V-ATPase) at the Golgi apparatus and trans-Golgi network (TGN). Oxr1 and Ncoa7 were predominantly localized at the Golgi and TGN membranes, dependent on their binding to various GTP-bound Rab proteins. In vitro experiments using purified recombinant proteins indicated that Oxr1 and Ncoa7 directly bind to the catalytic subunit of V-ATPase, inhibiting its ATP hydrolytic activity via their TLDc domains. We observed significant acidification of the Golgi/TGN lumen in Oxr1- and Ncoa7-depleted cells. Lectin blot analysis demonstrated that depletion of Oxr1 and Ncoa7 led to a defect in protein glycosylation, a major enzymatic posttranslational modification in the Golgi and TGN. Furthermore, depletion of Oxr1 and Ncoa7, along with drug-induced inhibition of glycosylation, increased lysosomal pH and sensitivity to silicon dioxide-induced membrane damage. This apparent lysosomal dysfunction suggested that, in addition to the Golgi and TGN, Oxr1 and Ncoa7 also contribute to the integrity of other organelles. Our findings indicate that Oxr1 and Ncoa7 protect the Golgi and TGN lumen from excess acidification by inhibiting V-ATPase activity and providing an optimal environment for enzymatic activity in the Golgi and TGN.