# Palmitoylation Code and Endosomal Sorting Regulate ABHD17A Plasma Membrane Targeting and Activity

## TL;DR

This study reveals how palmitoylation and endosomal sorting control the plasma membrane targeting and activity of the depalmitoylase ABHD17A.

## Contribution

The discovery of a palmitoylation code and YXXØ-dependent endosomal sorting mechanism regulating ABHD17A localization and function.

## Key findings

- N-terminal palmitoylation is essential for ABHD17A plasma membrane localization.
- Middle-region palmitoylation (C14, C15) is critical for PM targeting and catalytic activity.
- YXXØ motifs are required for endosomal sorting and surface abundance of ABHD17A.

## Abstract

Protein S-palmitoylation is a reversible lipid modification that regulates various aspects of protein function, including membrane association, subcellular localization, trafficking, stability, and activity. The depalmitoylase ABHD17A removes palmitate from multiple substrates, but its cellular positioning and the role of its own palmitoylation in regulating its function remain unclear. This study identifies a palmitoylation code within the conserved N-terminal cysteine cluster of ABHD17A, which governs its intracellular distribution and plasma membrane (PM) targeting. N-terminal palmitoylation is essential for PM localization. Through the use of code-restricted mutants, we found that modifications in the middle region (C14, C15) are critical for PM targeting and catalytic activity, while modifications at the front (C10, C11) and rear (C18) influence endosomal routing and delivery to the PM. Alanine scanning revealed that adjacent hydrophobic residues, particularly L9 and F13, are crucial for initial engagement with endomembranes. Sequence analysis and mutagenesis identified two tyrosine-based YXXØ motifs within the alpha/beta hydrolase fold; disruption of the proximal motif (L115A) decreased surface abundance and redirected ABHD17A to autophagosomes, indicating a need for YXXØ-dependent endosomal sorting, likely at the trans-Golgi network. Biochemical assays demonstrated a continuum of acylation states influenced by the palmitoylation code. This requirement for the middle region was conserved in ABHD17B and ABHD17C. Overall, our findings suggest a stepwise mechanism for ABHD17A delivery to the PM, enabling its depalmitoylase activity on membrane-bound substrates.