Oral Presentation International Congress on Neuronal Ceroid Lipofuscinoses 2025

Lysosomes perform many important cellular tasks including degradation of biomolecules, signalling and autophagy. In order to function, lysosomes contain soluble proteins that are transported to these organelles via lysosomal sorting receptors (LSR). To maintain efficient sorting of soluble lysosomal proteins, the LSRs are retrieved back to the trans-Golgi Network (TGN) for subsequent rounds of sorting. Rab7A recruitment of retromer, which in turn binds to the LSRs, is required for efficient retrieval.

CLN3, a glycosylated integral membrane protein localized to endolysosomes, is required for the efficient retromer/LSR interaction. In cells lacking or expressing non-functional CLN3, the LSRs are degraded in lysosomes, leading to decreased localization of soluble lysosomal proteins, and defective lysosomes. These cells are unable to efficiently degrade material that accumulates in lysosomes, disrupting several cellular functions including autophagy.

Palmitoylation is a reversible post-translational modification that can modulate protein localization, stability and protein-protein interactions. We have previously shown that the LSRs are palmitoylated and that modification is required for their efficient retrieval. We now demonstrate that CLN3 is palmitoylated on two cysteine residues, C339 and C340. Lack of palmitoylation at these sites had no impact on CLN3 stability but affected its localization. Compared to wild-type CLN3, there was less CLN3^C339A at endolysosomes, while CLN3^C340A had no effect. Using bioluminescence resonance energy transfer (BRET), we previously observed a decreased Rab7/retromer and retromer/LSR interaction in CLN3 knockout (CLN3^KO) HeLa cells. The overexpression of wild-type CLN3 and CLN3^C340A in CLN3^KO cells were able to restore these interactions, whereas CLN3^C339A could not.

We have shown that CLN3 is palmitoylated, and that this modification is required for the efficient endosome-to-TGN retrieval of the LSRs. Since the LSRs are also palmitoylated and that Rab7A palmitoylation is required for retromer recruitment, we propose that palmitoylation is used to establish an endosomal sorting domain.