title: An isoprenylation and palmitoylation motif promotes intraluminal vesicle delivery of proteins in cells from distant species.
abstract: The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (-CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms.
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Clara L
authors.initials: CL
authors.lastname: Oeste
authors.affiliation: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Pinar
authors.affiliation: Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, Oslo, Norway.
authors.forename: Kay O
authors.initials: KO
authors.lastname: Schink
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Martínez-Turrión
authors.affiliation: Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, Oslo, Norway.
authors.forename: Harald
authors.initials: H
authors.lastname: Stenmark
authors.affiliation: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Peñalva
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Pérez-Sala
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001233:Aspergillus nidulans / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D017124:Conserved Sequence; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D054878:Lipoylation; D008247:Lysosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D054876:Prenylation; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D022161:Transport Vesicles / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D020743:rhoB GTP-Binding Protein