ZDHHC7-mediated S-palmitoylation of ATG16L1 facilitates LC3 lipidation and autophagosome formation

ABSTRACT

Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12–ATG5 conjugate to form an ATG12–ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1^C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.

KEYWORDS:

• ATG16L1
• autophagosome
• autophagy
• LC3-II
• s-palmitoylation
• ZDHHC7

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Acknowledgments

We thank Prof. Feng Shao (National Institute of Biological Sciences, Beijing) and Prof. Yue Xu (Shanghai Jiao Tong University) for ATG16L1-KO HeLa cells stably expressing EGFP-LC3, Prof. Lifeng Pan (University of Chinese Academy of Sciences, Shanghai) for ATG16L1-KO HeLa cells, Prof. Masaki Fukata (National Institute for Physiological Sciences) for the expression vectors of the ZDHHC proteins, and Ms. Qin Deng (the Analytical and Testing Center of Chongqing University) for confocal technical support.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2024.2386915

Additional information

Funding

This study was supported by the National Natural Science Foundation of China (NSFC) (32170185 and 91854101), the Fundamental Research Funds for the Central Universities (2024CDJXY016, 2024CDJYXTD-010, SWU-KQ24036, 2022CDJYGRH-002), the Chongqing Postdoctoral Science Special Foundation, China (XmT2020194), the Chongqing Postdoctoral Science Foundation project (CSTB2023NSCQ-BHX0150) and the Venture Innovation Support Program for Chongqing Overseas Returnees (cx2022066).