RNF13 Dileucine Motif Variants L311S and L312P Interfere with Endosomal Localization and AP-3 Complex Association

Abstract

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

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“…It was also found that RNF13 associates with AP-3 [54][55][56], which mediates the transport from the tubular endosomes to the late endosomes and lysosomes and is therefore involved in the biogenesis of lysosome-related organelles [57,58]. Accordingly, our own work demonstrates that RNF13 interacts with the AP-3 complex in HEK293T cells [59], an interaction disrupted by an APbinding defective RNF13 mutant. By replacing the leucines with alanines in the di-leucine motif (i.e., L311A/L312A), the lysosomal localization of RNF13 was altered, suggesting that RNF13 s di-leucine sorting signal motif is crucial for the lysosomal targeting of the protein [59].…”

“…Accordingly, our own work demonstrates that RNF13 interacts with the AP-3 complex in HEK293T cells [59], an interaction disrupted by an APbinding defective RNF13 mutant. By replacing the leucines with alanines in the di-leucine motif (i.e., L311A/L312A), the lysosomal localization of RNF13 was altered, suggesting that RNF13 s di-leucine sorting signal motif is crucial for the lysosomal targeting of the protein [59]. Correspondingly, the knockdown of AP-3 altered the lysosomal localization of WT RNF13 and led to an alteration in the endosomal vesicle size [59].…”

“…Although this seminal study provided important evidence regarding the biological implication of expressing RNF13 variant L311S, it did not address the fact that the identified variants L311S and L312P completely altered RNF13's [D/E]xxxL[L/I]-type di-leucine sorting signal. In our own study, we demonstrated that both RNF13 L311S and L312P variants disrupted the association with AP-3 [59]. Notably, both RNF13 L311S and L312P variants no longer localized in the lysosomes, although they changed the morphology of the endolysosomal vesicles [59].…”

“…By replacing the leucines with alanines in the di-leucine motif (i.e., L311A/L312A), the lysosomal localization of RNF13 was altered, suggesting that RNF13 s di-leucine sorting signal motif is crucial for the lysosomal targeting of the protein [59]. Correspondingly, the knockdown of AP-3 altered the lysosomal localization of WT RNF13 and led to an alteration in the endosomal vesicle size [59]. Overall, this suggests that RNF13 might go into the endolysosomal compartments through an AP-3 dependent pathway.…”

“…Overall, this suggests that RNF13 might go into the endolysosomal compartments through an AP-3 dependent pathway. Future studies are critical to clarify the mechanism leading to the endolysosomal localization of RNF13 [59].…”