Lysosomal Targeting of Cystinosin Requires AP‐3 |
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Authors: | Zuzanna Andrzejewska Nathalie Névo Lucie Thomas Anne Bailleux Véronique Chauvet Alexandre Benmerah Corinne Antignac |
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Affiliation: | 1. Inserm U1163, Laboratory of Hereditary Kidney Diseases, Paris, France;2. Paris Descartes‐Sorbonne Paris Cité University, Imagine Institute, Paris, France;3. Assistance Publique ‐ H?pitaux de Paris (AP‐HP), Department of Genetics, Necker Hospital, Paris,, France |
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Abstract: | Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine‐based signal (GYDQL) in its C‐terminal tail and a non‐classical motif in its fifth inter‐TM loop. Using the yeast two‐hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP‐3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP‐3‐depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP‐3 knockdown cells where it also accumulated in the trans‐Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63‐GYDQL chimeric protein were not increased when clathrin‐mediated endocytosis was impaired, our data show that the tyrosine‐based motif of cystinosin is a ‘strong’ AP‐3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway. |
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Keywords: | adaptor protein complex cystinosin cystinosis lysosomal storage disorder lysosomal targeting transmembrane protein tyrosine‐based motif |
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