title: Identification and subcellular localization of a new cystinosin isoform.
abstract: Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [(35)S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.
authors.affiliation: Department of Nephrology and Urology, Division of Nephrology, Ospedale Bambino Gesù Children's Hospital and Research Insitute, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Taranta
authors.forename: Stefania
authors.initials: S
authors.lastname: Petrini
authors.forename: Alessia
authors.initials: A
authors.lastname: Palma
authors.forename: Liliana
authors.initials: L
authors.lastname: Mannucci
authors.forename: Martijn J
authors.initials: MJ
authors.lastname: Wilmer
authors.forename: Veronica
authors.initials: V
authors.lastname: De Luca
authors.forename: Francesca
authors.initials: F
authors.lastname: Diomedi-Camassei
authors.forename: Serena
authors.initials: S
authors.lastname: Corallini
authors.forename: Francesco
authors.initials: F
authors.lastname: Bellomo
authors.forename: Lambert P
authors.initials: LP
authors.lastname: van den Heuvel
authors.forename: Elena N
authors.initials: EN
authors.lastname: Levtchenko
authors.forename: Francesco
authors.initials: F
authors.lastname: Emma
mesh_terms: D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003553:Cystine / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005091:Exons / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007536:Isomerism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
chemical_list: D026921:Amino Acid Transport Systems, Neutral; C111445:CTNS protein, human; D018076:DNA, Complementary; D003553:Cystine