title: Lysosomal targeting of the CLN7 membrane glycoprotein and transport via the plasma membrane require a dileucine motif.
abstract: CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N- and C-terminal tails of CLN7 in the cytosol and two N-glycosylation sites at N371 and N376. Both partially and non-glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4-chimera fused to the N- and C-terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine-based motif in the N-terminus and two tandem tyrosine-based signals in the C-terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine-based motif is of critical importance for lysosomal localization of the full-length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co-expression of dominant-negative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N-terminal dileucine-based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin-mediated endocytosis of CLN7.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany.
authors.forename: Pieter
authors.initials: P
authors.lastname: Steenhuis
authors.forename: Stephanie
authors.initials: S
authors.lastname: Herder
authors.forename: Suyin
authors.initials: S
authors.lastname: Gelis
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.forename: Stephan
authors.initials: S
authors.lastname: Storch
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D019921:Biotinylation; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007930:Leucine; D016464:Lysosomal Storage Diseases / Q000235:genetics / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D002966:Clathrin; D051907:Lysosomal Membrane Proteins; C522398:MFSD8 protein, human; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D007930:Leucine