# Regulation of V-ATPases by Phosphoinositides

> **NIH NIH R01** · UPSTATE MEDICAL UNIVERSITY · 2020 · $275,400

## Abstract

PROJECT SUMMARY
 V-ATPases are highly conserved, multisubunit proton pumps that acidify organelles including
lysosomes, endosomes, Golgi apparatus, and secretory granules in all eukaryotic cells. Each of these
organelles requires tuning of its luminal pH to a narrow range for its function. However, it is not fully
understood how these pH ranges are maintained or how V-ATPases in different organelles respond when
environmental conditions challenge local pH gradients. Our central hypothesis is that PI lipids provide
organelle-specific inputs to locally regulate V-ATPase activity, and thus can impact individual subsets of the
many functions dependent on V-ATPase activity and organelle acidification. In Aim 1, we use the well-
characterized yeast V-ATPase system to characterize binding of the cytosolic domains of the membrane-
bound a-subunit isoforms, Vph1 and Stv1, to phosphoinositide (PI) lipids. We have found that vacuole-resident
Vph1 is able to bind the vacuolar signaling lipid PI(3,5)P2 and Golgi-resident Stv1 prefers the Golgi-enriched
lipid PI(4)P. We will quantify and characterize these binding preferences and probe how binding of specific
lipids activates V-ATPases containing individual a-subunit isoforms. Based on recent structures and homology
modeling, we will then develop lipid-binding mutants for each isoform. Finally, we will evaluate PI binding
specificities of the four human a-subunit isoforms. We have tested three of the four human isoforms and found
evidence of PI-specific interactions in vitro. We will characterize specificity and affinities of PI binding and
identify potential binding sites. In Aim 2, we address the spatial and temporal consequences of PI lipid binding
to intact V-ATPases containing full-length Vph1 and Stv1. We hypothesize that PI lipids exert spatial control by
activating V-ATPases in their organelles of residence. Specifically, we propose that: 1) PI(3,5)P2 binding to
Vph1-containing V-ATPases induces a conformation that stabilizes interactions between the peripheral V1
sector and the integral membrane Vo sector of the V-ATPase, increasing activity, 2) PI(4)P binding to Stv1-
containing V-ATPases promotes retention in the Golgi apparatus, and 3) PI(3)P promotes acidification of the
endocytic pathway by interacting with Vph1-containing V-ATPases. In contrast, hyperosmotic stress promotes
a rapid and transient rise in PI(3,5)P2 that reversibly activates Vph1-containing V-ATPases in the vacuole. We
will analyze the mechanisms of this temporal V-ATPase activation and its role in providing short-term
adaptation to salt stress. These experiments link PI and V-ATPase isoform content, two fundamental features
of organelle identity. They promise to provide novel insights into the many diseases linked to defective
organelle acidification, including neurodegeneration, cancer, and mycobacterial infection, and could suggest
new therapeutic routes.

## Key facts

- **NIH application ID:** 9933083
- **Project number:** 5R01GM126020-03
- **Recipient organization:** UPSTATE MEDICAL UNIVERSITY
- **Principal Investigator:** PATRICIA M KANE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $275,400
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9933083

## Citation

> US National Institutes of Health, RePORTER application 9933083, Regulation of V-ATPases by Phosphoinositides (5R01GM126020-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9933083. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*