A Genetic Screen for Investigating the Human Lysosomal CystineTransporter, Cystinosin

Scientific Reports
Anup Arunrao DeshpandeAnand Kumar Bachhawat

Abstract

Cystinosin, a lysosomal transporter is involved in the efflux of cystine from the lysosome to the cytosol. Mutations in the human cystinosin gene (CTNS) cause cystinosis, a recessive autosomal disorder. Studies on cystinosin have been limited by the absence of a robust genetic screen. In the present study we have developed a dual strategy for evaluating cystinosin function that is amenable to rapid genetic analysis. We show that human cystinosin expressed in this yeast confers growth on cystine when the protein is mistargeted to the plasma membrane by the deletion of the C-terminal targeting signal, GYQDL. We also screened a vacuolar protein sorting deletion library, and subsequently created multiple vps deletion mutants for kinetic studies. The double deletion, vps1Δvps17Δ, greatly enhanced uptake. This enabled validation by kinetic studies, including first studies on the WT CTNS protein (that contained the GYQDL motif). Using this screen we isolated several gain of function mutants, G131S/D, G309S/D, A137V, G197R, S270T, L274F and S312N showing enhanced growth on low concentrations of cystine. Kinetic analysis yielded insights into the role of the residues (including one of the patient mutations, G197R). The results indicate ...Continue Reading

References

Oct 1, 1990·The Journal of Cell Biology·L A SilveiraR Schekman
May 12, 2005·The FEBS Journal·Xiao-Dong GaoNeta Dean
Jul 8, 2009·Genetic Testing and Molecular Biomarkers·Sha TangTaosheng Huang
Apr 22, 2011·The Journal of Biological Chemistry·Amit Kumar Yadav, Anand Kumar Bachhawat
Nov 22, 2012·Proceedings of the National Academy of Sciences of the United States of America·Adrien JézégouBruno Gasnier
Feb 7, 2013·Journal of Biosciences·Jacob HaydenKyoungtae Kim
Mar 11, 2015·Traffic·Zuzanna AndrzejewskaCorinne Antignac

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