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2.
Nat Commun ; 9(1): 792, 2018 02 23.
Article in English | MEDLINE | ID: mdl-29476049

ABSTRACT

Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking.


Subject(s)
Endosomes/metabolism , Integrin beta1/metabolism , Vesicular Transport Proteins/metabolism , Cell Adhesion , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Movement , Endosomes/genetics , HeLa Cells , Humans , Integrin beta1/genetics , Protein Transport , Vesicular Transport Proteins/genetics
3.
EMBO Rep ; 17(6): 800-10, 2016 06.
Article in English | MEDLINE | ID: mdl-27113756

ABSTRACT

The oxysterol-binding protein (OSBP)-related proteins ORP5 and ORP8 have been shown recently to transport phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) at ER-PM contact sites. PS is also transferred from the ER to mitochondria where it acts as precursor for mitochondrial PE synthesis. Here, we show that, in addition to ER-PM contact sites, ORP5 and ORP8 are also localized to ER-mitochondria contacts and interact with the outer mitochondrial membrane protein PTPIP51. A functional lipid transfer (ORD) domain was required for this localization. Interestingly, ORP5 and ORP8 depletion leads to defects in mitochondria morphology and respiratory function.


Subject(s)
Endoplasmic Reticulum/metabolism , Mitochondria/metabolism , Receptors, Steroid/metabolism , Cell Line , Endoplasmic Reticulum/ultrastructure , Gene Knockdown Techniques , Humans , Lipid Metabolism , Mitochondria/genetics , Mitochondria/ultrastructure , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Receptors, Steroid/chemistry , Receptors, Steroid/genetics
4.
Traffic ; 16(12): 1288-305, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26403612

ABSTRACT

Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA-gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal-lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal-lysosomal fusion events.


Subject(s)
Endocytosis/physiology , Endosomes/metabolism , Lysosomes/metabolism , Vesicular Transport Proteins/metabolism , Endosomes/ultrastructure , Gene Knockdown Techniques , HeLa Cells , Humans , Lysosomes/ultrastructure , Membrane Fusion/physiology , Microscopy, Electron , Microscopy, Fluorescence , Protein Transport , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics
5.
J Cell Sci ; 126(Pt 15): 3409-16, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23750006

ABSTRACT

Targeting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) in polarized epithelial cells depends on their association with detergent-resistant membrane microdomains called rafts. In MDCK cells, GPI-APs associate with rafts in the trans-Golgi network and are directly delivered to the apical membrane. It has been shown that oligomerization is required for their stabilization in rafts and their apical targeting. In hepatocytes, GPI-APs are first delivered to the basolateral membrane and secondarily reach the apical membrane by transcytosis. We investigated whether oligomerization is required for raft association and apical sorting of GPI-APs in polarized HepG2 cells, and at which step of the pathway oligomerization occurs. Model proteins were wild-type GFP-GPI and a double cysteine GFP-GPI mutant, in which GFP dimerization was impaired. Unlike wild-type GFP-GPI, which was efficiently endocytosed and transcytosed to the apical surface, the double cysteine mutant was basolaterally internalized, but massively accumulated in early endosomes, and reached the bile canaliculi with delayed kinetics. The double cysteine mutant was less resistant to Triton X-100 extraction, and formed fewer high molecular weight complexes. We conclude from these results that, in hepatocytes, oligomerization plays a key role in targeting GPI-APs to the apical membrane, by increasing their affinity for rafts and allowing their transcytosis.


Subject(s)
GPI-Linked Proteins/metabolism , Hepatocytes/metabolism , Cell Growth Processes/physiology , Cell Polarity/physiology , Cysteine , Endocytosis/physiology , GPI-Linked Proteins/genetics , Hep G2 Cells , Hepatocytes/cytology , Humans , Protein Transport , Transcytosis
6.
Hum Mutat ; 33(12): 1656-64, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22753090

ABSTRACT

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apical-basolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotype-phenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR.


Subject(s)
Arthrogryposis/diagnosis , Arthrogryposis/genetics , Carrier Proteins/genetics , Cholestasis/diagnosis , Cholestasis/genetics , Genetic Association Studies , Renal Insufficiency/diagnosis , Renal Insufficiency/genetics , Vesicular Transport Proteins/genetics , Child, Preschool , Female , HEK293 Cells , Heterozygote , Humans , Male , Models, Molecular , Molecular Diagnostic Techniques , Protein Transport , RNA Splice Sites , Sequence Analysis, DNA
7.
Mol Biol Cell ; 20(17): 3792-800, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19605558

ABSTRACT

In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2-positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.


Subject(s)
Cell Polarity , Clathrin/metabolism , Endocytosis/physiology , Glycosylphosphatidylinositols/metabolism , Hepatocytes/cytology , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , CD13 Antigens/genetics , CD13 Antigens/metabolism , CD59 Antigens/genetics , CD59 Antigens/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Line , Clathrin/genetics , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Dynamins/genetics , Dynamins/metabolism , Glycosylphosphatidylinositols/genetics , Hepatocytes/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Transport/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism
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