Abstract
Cystinosin mediates an ATP-dependent cystine efflux from lysosomes and causes, if mutated, nephropathic cystinosis, a rare inherited lysosomal storage disease. Alternative splicing of the last exon of the cystinosin sequence produces the cystinosin-LKG isoform that is characterized by a different C-terminal region causing changes in the subcellular distribution of the protein. We have constructed RFP-tagged proteins and demonstrated by site-directed mutagenesis that the carboxyl-terminal SSLKG sequence of cystinosin-LKG is an important sorting motif that is required for efficient targeting the protein to the plasma membrane, where it can mediate H+ coupled cystine transport. Deletion of the SSLKG sequence reduced cystinosin-LKG expression in the plasma membrane and cystine transport by approximately 30%, and induced significant accumulation of the protein in the Golgi apparatus and in lysosomes. Cystinosin-LKG, unlike the canonical isoform, also moves to the lysosomes by the indirect pathway, after endocytic retrieval from the plasma membrane, mainly by a clathrin-mediated endocytosis. Nevertheless, silencing of AP-2 triggers the clathrin-independent endocytosis, showing the complex adaptability of cystinosin-LKG trafficking.
Highlights
The carboxyl-terminal portion of proteins often contains key sequences that are essential for accurate protein sorting and signaling [1,2]
In addition to the originally described lysosomal protein, we have identified a second isoform (UniProt # O60931-2), termed cystinosin-LKG, based on the sequence of the last amino acids, that is produced by an alternative splicing of exon 12, and which differs from its canonical counterpart only in the carboxyl-terminal sequence (Fig 1)
Co-localization with endoplasmic reticulum (ER) markers was less prominent and no significant changes were observed between wild-type and ΔSSLKG mutants (RrLKG = 0.40 ± 0.03 vs RrΔSSLKG = 0.42 ± 0.02; p = NS)
Summary
The carboxyl-terminal portion of proteins often contains key sequences that are essential for accurate protein sorting and signaling [1,2]. Proteins can undergo reversible or irreversible post-translational modifications, producing additional changes at their terminal sequences that modify their biological properties. Changes in the C-terminal sequence can modify temporal and/or spatial distribution of peptides in cells, which often translates into different biological properties of a given protein, according to the cell compartment in which it is expressed, and to the biological state of the cell [3]. The CTNS gene encodes for cystinosin (UniProt # O60931-1), a cystine/H+ symporter that mediates the efflux of cystine in the presence of a proton gradient. Essential to the role of cystinosin in cells, sorting of the protein to lysosomes requires at least two targeting motifs, namely a classical tyrosine-based motif (GYDQL), located at the C-terminal end, and a conformational motif (YFPQA), located in the putative fifth inter-transmembrane loop [6]
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