title: A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer.
abstract: The mammalian retromer is a multimeric protein complex involved in mediating endosome-to-trans-Golgi-network retrograde transport of the cation-independent mannose-6-phosphate receptor. The retromer is composed of two subcomplexes, one containing SNX1 and forming a membrane-bound coat, the other comprising VPS26, VPS29 and VPS35 and being cargo-selective. In yeast, an additional sorting nexin--Vps17p--is a component of the membrane bound coat. It remains unclear whether the mammalian retromer requires a functional equivalent of Vps17p. Here, we have used an RNAi loss-of-function screen to examine whether any of the other 30 mammalian sorting nexins are required for retromer-mediated endosome-to-trans-Golgi-network retrieval of the cation-independent mannose-6-phosphate receptor. Using this screen, we identified two proteins, SNX5 and SNX6, that, when suppressed, induced a phenotype similar to that observed upon suppression of known retromer components. Whereas SNX5 and SNX6 colocalised with SNX1 on early endosomes, in immunoprecipitation experiments only SNX6 appeared to exist in a complex with SNX1. Interestingly, suppression of SNX5 and/or SNX6 resulted in a significant loss of SNX1, an effect that seemed to result from post-translational regulation of the SNX1 level. Such data suggest that SNX1 and SNX6 exist in a stable, endosomally associated complex that is required for retromer-mediated retrieval of the cation-independent mannose-6-phosphate receptor. SNX5 and SNX6 may therefore constitute functional equivalents of Vps17p in mammals.
authors.affiliation: The Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wassmer
authors.forename: Naomi
authors.initials: N
authors.lastname: Attar
authors.forename: Miriam V
authors.initials: MV
authors.lastname: Bujny
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Oakley
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Traer
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cullen
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D005820:Genetic Testing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D021381:Protein Transport / Q000502:physiology*; D034622:RNA Interference; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C513978:SNX1 protein, human; C402667:SNX5 protein, human; C431150:SNX6 protein, human; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins