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PLoS One
2014 Jan 01;99:e107190. doi: 10.1371/journal.pone.0107190.
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An isoprenylation and palmitoylation motif promotes intraluminal vesicle delivery of proteins in cells from distant species.
Oeste CL
,
Pinar M
,
Schink KO
,
Martínez-Turrión J
,
Stenmark H
,
Peñalva MA
,
Pérez-Sala D
.
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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.
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25207810
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Figure 2. Endolysosomal localization of CINCCKVL-chimeric proteins depends on posttranslational processing.(A) HeLa cells were transiently transfected with the indicated constructs and 24 h later they were cultured in serum-free medium for another 24 h. Cell lysis and fractionation were achieved as described in the Experimental section. Upper panels show the amount of the constructs in total cell lysates and lower panels depict the levels of the constructs in S100 (soluble) and P100 (particulate) fractions, assessed by western blot with an anti-GFP antibody. Hsp90 was used as a loading control, RhoGDI as a marker of the soluble fraction and vimentin as a particulate fraction marker. Results are representative from three experiments with virtually identical results. The positions of the 25 and 100 kDa markers are shown for reference. (B) HeLa cells were transfected with GFP-8, its palmitoylation defective mutant (GFP-8-C240, 243S), its isoprenylation defective mutant (GFP-8-C244S) or GFP, as indicated, and stained with LTR prior to live confocal microscopy imaging. (C) GFP-8-transfected HeLa cells were treated with 20 µM 2-bromopalmitate for 6 h or 10 µM simvastatin for 24 h in serum-free medium and stained with LTR before live observation by confocal microscopy. (D) BAEC were transfected with Dendra-8 and treated with simvastatin for 24 h (left panel) prior to eliciting a green-to-red photoswitch using UV light (middle panel). Immediately after photoswitching simvastatin was removed and the localization of the red, photoconverted protein was assessed 24 h later (right panel). Scale bar, 10 µm.
Figure 3. Effects of agents modulating cholesterol synthesis and traffic on the distribution of CINCCKVL constructs.(A) HeLa cells were transfected with GFP-8 (upper panels) or GFP-CD63 (lower panels). 24 h later cells were treated with 100 µM ZGA or 10 µM U18666A for 24 h and stained with LTR as above. Insets show enlarged areas of interest. The single channels corresponding to the areas in insets are shown below each image. (B) BAEC were transfected with GFP-8 and treated with ZGA or U18666A as described in (A). Insets show enlarged areas of interest and lower panels depict fluorescence intensity profiles along a section (see dotted lines marked by asterisks).
Figure 4. C-terminal sequences of CINCCKVL-chimeric proteins, RhoB homologs and related proteins from diverse species.The C-terminal sequences of GFP-8 and tRFP-T-8 are shown on top, together with a schematic view of the lipidic modifications; palmitates are shown in black and the geranylgeranyl moiety in blue. The Pubmed accession numbers of genes coding for proteins bearing C-terminal sequences similar to that of RhoB are shown in the lower panel. Potential sites for palmitoylation are shown in red and the isoprenylation cysteine in green.
Figure 5. Localization of RhoB-related proteins in amphibian cells.(A) Xenopus laevis RhoB (GFP-RhoB X) or GFP-8 were expressed in Xenopus laevis A6 cells and acidic compartments were stained with LTR. The right panel depicts the extent of co-localization of GFP and LTR signals shown as Pearson coefficients (x100) or co-localization rates (in percentages). Results are average values of at least 30 cells per condition ± standard error of mean (SEM). *p<1×10−6
vs GFP-8 by Student’s t-test. (B) Effect of agents altering lysosomal function on the distribution of tRFP-T-8. Xenopus A6 cells were co-transfected with Lamp1-GFP to mark lysosomes and tRFP-T-8, and treated with 10 µM U18666A or 10 µM chloroquine, as indicated. (C) Dependence of tRFP-T-8 localization on posttranslational modification in Xenopus cells. A6 cells were transfected with tRFP-T or tRFP-T-8 and treated with 10 µM simvastatin or 20 µM 2-bromopalmitate and imaged live by confocal microscopy.
Figure 6. Localization of tRFP-T-8 in High Five insect cells.High Five cells were transfected with Lamp1-GFP and tRFP-T-8 and live cells were visualized by confocal microscopy. The overlays of single fluorescent z-sections alone and with the Differential Interference Contrast image are shown.
Figure 7. Localization of GFP-8 in Aspergillus nidulans.Strains of Aspergillus nidulans expressing GFP-8 or its palmitoylation deficient mutant (GFP-8-C240, 243S) were imaged as described in Methods. (A and B) show co-localization of the constructs with the vacuole marker, CMAC. Insets show grayscale images for better contrast. (C) Kymograph showing the movements of various GFP-8-positive compartments. Rapidly moving endosomes (likely corresponding to early endosomes) are marked by arrowheads (red), static vesicles (likely corresponding to vacuoles) are marked by arrows and potential points of contact between endosomes and vacuoles are depicted by asterisks. (D) Graph representing velocities of individual endosomes.
Figure 1. Localization of CINCCKVL chimeras in human cells.(A) Human fibroblasts were transfected with the indicated constructs and observed live by confocal microscopy after 16 h in serum-depleted medium. To the right of every condition, zoom-ins of original pictures and fluorescence intensity profiles along a section (see dotted lines marked by asterisks) are shown. The magnification used for zoom-ins is seven-fold with respect to the corresponding full-sized images. F.I., fluorescence intensity. (B) HeLa cells were transiently transfected with the indicated constructs and treated with 25 nM LTR for 15 min, where indicated, prior to live confocal microscopy observation. Bottom panels show cells that were treated with 10 µM chloroquine 24 h post-transfection for a further 24 h. Insets show details of the pictured cells. (C) Cells transfected as in (B) were visualized live by super-resolution microscopy (SIM). Insets show individual MVB and their ILV decorated with CINCCKVL fluorescent proteins. Scale bar, 20 µm.
Abenza,
Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement.
2012, Pubmed
Abenza,
Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement.
2012,
Pubmed
Adamson,
Intracellular localization of the P21rho proteins.
1992,
Pubmed
Aicart-Ramos,
Protein palmitoylation and subcellular trafficking.
2011,
Pubmed
Amiya,
Angiotensin II impairs endothelial nitric-oxide synthase bioavailability under free cholesterol-enriched conditions via intracellular free cholesterol-rich membrane microdomains.
2013,
Pubmed
Bonifacino,
Signals for sorting of transmembrane proteins to endosomes and lysosomes.
2003,
Pubmed
Braulke,
Sorting of lysosomal proteins.
2009,
Pubmed
Bucci,
The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.
1992,
Pubmed
Canto,
Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.
2013,
Pubmed
Coutinho,
A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.
2012,
Pubmed
Dores,
ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.
2012,
Pubmed
Elias,
Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases.
2012,
Pubmed
Flannery,
Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes.
2010,
Pubmed
Fortwendel,
Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus.
2012,
Pubmed
Gharbi,
Study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin PGA1: focus on vimentin.
2007,
Pubmed
Ghosh,
Mannose 6-phosphate receptors: new twists in the tale.
2003,
Pubmed
Gilk,
Bacterial colonization of host cells in the absence of cholesterol.
2013,
Pubmed
Hirata,
Genes that cause aberrant cell morphology by overexpression in fission yeast: a role of a small GTP-binding protein Rho2 in cell morphogenesis.
1998,
Pubmed
Huotari,
Endosome maturation.
2011,
Pubmed
Klöpper,
Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis.
2012,
Pubmed
Kneen,
Green fluorescent protein as a noninvasive intracellular pH indicator.
1998,
Pubmed
Koga,
A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.
2011,
Pubmed
Lambou,
Fungi have three tetraspanin families with distinct functions.
2008,
Pubmed
Leung,
Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage.
2008,
Pubmed
Ma,
Rho2 is a target of the farnesyltransferase Cpp1 and acts upstream of Pmk1 mitogen-activated protein kinase signaling in fission yeast.
2006,
Pubmed
Matsuo,
Role of LBPA and Alix in multivesicular liposome formation and endosome organization.
2004,
Pubmed
McCormick,
Palmitoylation controls recycling in lysosomal sorting and trafficking.
2008,
Pubmed
Michaelson,
Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding.
2001,
Pubmed
Möbius,
Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway.
2003,
Pubmed
Nickerson,
A concentric circle model of multivesicular body cargo sorting.
2007,
Pubmed
Oeste,
Interactions between autophagic and endo-lysosomal markers in endothelial cells.
2013,
Pubmed
Pantazopoulou,
Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis.
2009,
Pubmed
Peng,
Palmitoylation plays a role in targeting Vac8p to specific membrane subdomains.
2006,
Pubmed
Perez-Sala,
Protein isoprenylation in biology and disease: general overview and perspectives from studies with genetically engineered animals.
2007,
Pubmed
Platta,
Endocytosis and signaling.
2011,
Pubmed
Pérez-Sala,
The C-terminal sequence of RhoB directs protein degradation through an endo-lysosomal pathway.
2009,
Pubmed
Raiborg,
The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins.
2009,
Pubmed
Rink,
Rab conversion as a mechanism of progression from early to late endosomes.
2005,
Pubmed
Roberts,
Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification.
2008,
Pubmed
Roth,
Global analysis of protein palmitoylation in yeast.
2006,
Pubmed
Sahu,
Microautophagy of cytosolic proteins by late endosomes.
2011,
Pubmed
Schweizer,
Cysteine34 of the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting.
1996,
Pubmed
Shaner,
Improving the photostability of bright monomeric orange and red fluorescent proteins.
2008,
Pubmed
Sobo,
Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking.
2007,
Pubmed
Stamatakis,
Isoprenylation of RhoB is necessary for its degradation. A novel determinant in the complex regulation of RhoB expression by the mevalonate pathway.
2002,
Pubmed
Stipp,
Functional domains in tetraspanin proteins.
2003,
Pubmed
Toulmay,
Direct imaging reveals stable, micrometer-scale lipid domains that segregate proteins in live cells.
2013,
Pubmed
Ullrich,
Rab11 regulates recycling through the pericentriolar recycling endosome.
1996,
Pubmed
Valero,
Structural determinants allowing endolysosomal sorting and degradation of endosomal GTPases.
2010,
Pubmed
Veit,
Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25.
1996,
Pubmed
Wang,
Palmitoylated cysteine 192 is required for RhoB tumor-suppressive and apoptotic activities.
2005,
Pubmed
Williams,
The emerging shape of the ESCRT machinery.
2007,
Pubmed