# NoxO1 promotes endosome formation and reduces intracellular vesicle processing

## TL;DR

NoxO1 promotes the formation of endosomes and lysosomes, slows down the processing of cellular cargo, and activates a key regulator of lysosomal biogenesis.

## Contribution

NoxO1's ROS-independent role in regulating endosomal trafficking and lysosomal biogenesis via Erbin and TFEB is revealed.

## Key findings

- NoxO1 overexpression increases early endosome and lysosome abundance in cultured cells.
- NoxO1 activates TFEB via Erbin, expanding the lysosomal/CLEAR program.
- NoxO1 slows endosomal trafficking and increases lysosomal cargo retention.

## Abstract

NADPH oxidase organizer 1 (NoxO1) is known as a scaffold cytoplasmic subunit of the reactive oxygen species (ROS) forming Nox1 complex. We previously identified an interaction between NoxO1 and Erbin, a cytosolic scaffold protein that associates with Epidermal Growth Factor Receptor (EGFR), but its ROS-independent roles remain poorly understood.

Here, we demonstrate that NoxO1 overexpression remodels the endolysosomal system by expanding early endosomes and lysosomes. A calibrated six-compartment ordinary differential equation model of EGFR trafficking predicts a slowed down intracellular trafficking: NoxO1 overexpression increased internalization rates by 14 % while reducing degradative sorting by 48 %, lysosomal transfer by 24 %, and final degradation by 41 %. Using fluorescent cargo (EGF and BSA), we confirmed enhanced internalization and cargo accumulation in lysosomes, supporting the idea of prolonged lysosomal retention in NoxO1 overexpressing cells. Mechanistically, NoxO1 activated transcription factor EB (TFEB), the master regulator of lysosomal biogenesis, in an Erbin-dependent but ROS independent manner. Proximity ligation assays revealed spatial association of NoxO1, Erbin, EGFR, and TFEB, suggesting a multi-protein regulatory complex. Genetic ablation of Erbin abolished NoxO1-induced increases in early endosome (EEA1) and lysosome (LAMP1) markers, confirming Erbin's essential role.

In conclusion, via its interaction with Erbin NoxO1 promotes activation of TFEB, contributes to lysosome formation while delaying cargo degradation.

Image 1

•NoxO1 increases early endosome and lysosome abundance in cultured cells.•NoxO1 slows endosomal trafficking and increases lysosomal cargo retention.•NoxO1 activates TFEB via Erbin, expanding the lysosomal/CLEAR program.•Minimal contribution of NOX1-derived ROS under the tested conditions.•A calibrated trafficking model explains the observed compartment shifts.

NoxO1 increases early endosome and lysosome abundance in cultured cells.

NoxO1 slows endosomal trafficking and increases lysosomal cargo retention.

NoxO1 activates TFEB via Erbin, expanding the lysosomal/CLEAR program.

Minimal contribution of NOX1-derived ROS under the tested conditions.

A calibrated trafficking model explains the observed compartment shifts.