title: Expression and subcellular distribution of gephyrin in non-neuronal tissues and cells.
abstract: Gephyrin is a scaffolding protein required for the accumulation of inhibitory neurotransmitter receptors at neuronal postsynaptic membranes. In non-neuronal tissues, gephyrin is indispensible for the biosynthesis of molybdenum cofactor, the prosthetic group of oxidoreductases including sulfite oxidase and xanthine oxidase. However, the molecular and cellular basis of gephyrin's non-neuronal function is poorly understood; in particular, the roles of its splice variants remain enigmatic. Here, we used cDNA screening as well as Northern and immunoblot analyses to show that mammalian liver contains only a limited number of gephyrin splice variants, with the C3-containing variant being the predominant isoform. Using new and established anti-gephyrin antibodies in immunofluorescence and subcellular fractionation studies, we report that gephyrin localizes to the cytoplasm of both tissue hepatocytes and cultured immortalized cells. These findings were corroborated by RNA interference studies in which the cytosolic distribution was found to be abolished. Finally, by blue-native PAGE we show that cytoplasmic gephyrin is part of a ~600 kDa protein complex of yet unknown composition. Our data suggest that the expression pattern of non-neuronal gephyrin is simpler than indicated by previous evidence. In addition, gephyrin's presence in a cytosolic 600 kDa protein complex suggests that its metabolic and/or other non-neuronal functions are exerted in the cytoplasm and are not confined to a particular subcellular compartment.
authors.affiliation: Department of Medical Cell Biology, Institute for Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany. nawrotzki@ana.uni-heidelberg.de
authors.forename: Ralph
authors.initials: R
authors.lastname: Nawrotzki
authors.forename: Markus
authors.initials: M
authors.lastname: Islinger
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Vogel
authors.forename: Alfred
authors.initials: A
authors.lastname: Völkl
authors.forename: Joachim
authors.initials: J
authors.lastname: Kirsch
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons; D009928:Organ Specificity; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C073441:gephyrin