Two dileucine motifs mediate late endosomal/lysosomal targeting of “Transmembrane protein 192” (TMEM192) and a C-terminal cysteine is responsible for disulphide bond formation in TMEM192 homodimers

Overview & Synopsis

“Transmembrane protein 192” (TMEM192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and unknown function that was initially discovered by organellar proteomics. Subsequently, localisation in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192 and homodimers of TMEM192 linked by disulphide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucines resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulphide bond formation, mutagenesis of cysteine residues was performed. Mutation of the cysteine residue C266 abolished disulphide bridge formation between TMEM192 molecules indicating that TMEM192 dimers are linked by a disulphide bridge between their C-terminal tails. According to the predicted topology, C266 would be localised in the reductive milieu of the cytosol where disulphide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays the localisation of N- and C-terminus of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.