Comparative proximity biotinylation implicates RAB18 in sterol mobilization and biosynthesis

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18-interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. 12 of these 28 interactions are supported by prior reports and we have directly validated novel interactions with SEC22A, TMCO4 and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP (emopamil binding protein) is a Δ8-Δ7 sterol isomerase and OSBPL2/ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We find that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Further, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

[1]  S. Eimer,et al.  RAB18 loss interferes with lipid droplet catabolism and provokes autophagy network adaptations. , 2019, Journal of molecular biology.

[2]  J. Bonifacino,et al.  Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling , 2019, Nature Cell Biology.

[3]  F. Barr,et al.  Rab regulation by GEFs and GAPs during membrane traffic. , 2019, Current opinion in cell biology.

[4]  I. Belevich,et al.  REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis , 2019, Molecular biology of the cell.

[5]  J. Shendure,et al.  Mutations in the translocon‐associated protein complex subunit SSR3 cause a novel congenital disorder of glycosylation , 2019, Journal of inherited metabolic disease.

[6]  Yan G Zhao,et al.  The ER-Localized Protein DFCP1 Modulates ER-Lipid Droplet Contact Formation. , 2019, Cell reports.

[7]  S. Munro,et al.  In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation , 2019, bioRxiv.

[8]  R. Parton,et al.  Crystal structure of ORP2-ORD in complex with PI(4,5)P2 , 2019 .

[9]  Martin Eisenacher,et al.  The PRIDE database and related tools and resources in 2019: improving support for quantification data , 2018, Nucleic Acids Res..

[10]  B. Delprat,et al.  ER-mitochondria cross-talk is regulated by the Ca2+ sensor NCS1 and is impaired in Wolfram syndrome , 2018, Science Signaling.

[11]  J. Clayton-Smith,et al.  Lathosterolosis: A Relatively Mild Case with Cataracts and Learning Difficulties. , 2018, JIMD reports.

[12]  Pedro Carvalho,et al.  Here, there, and everywhere: The importance of ER membrane contact sites , 2018, Science.

[13]  N. Perrimon,et al.  Efficient proximity labeling in living cells and organisms with TurboID , 2018, Nature Biotechnology.

[14]  B. Antonny,et al.  The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol. , 2018, Annual review of biochemistry.

[15]  A. Harada,et al.  The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution , 2018, The Journal of cell biology.

[16]  Xiaonan Liu,et al.  An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations , 2018, Nature Communications.

[17]  Peng Li,et al.  Rab18 promotes lipid droplet (LD) growth by tethering the ER to LDs through SNARE and NRZ interactions , 2018, The Journal of cell biology.

[18]  Kelly M. Hines,et al.  Prevention of Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome , 2018, Scientific Reports.

[19]  B. Burke,et al.  BioID: A Screen for Protein‐Protein Interactions , 2018, Current protocols in protein science.

[20]  C. Ungermann,et al.  A guanine nucleotide exchange factor (GEF) limits Rab GTPase–driven membrane fusion , 2017, The Journal of Biological Chemistry.

[21]  S. Eimer,et al.  Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy , 2017, The EMBO journal.

[22]  Edward S Boyden,et al.  Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy , 2017, The EMBO journal.

[23]  Ke Xu,et al.  NuMA recruits dynein activity to microtubule minus-ends at mitosis , 2017, eLife.

[24]  W. Hong,et al.  SNARE proteins in membrane trafficking , 2017, Traffic.

[25]  M. Imperiale,et al.  Identification of Rab18 as an Essential Host Factor for BK Polyomavirus Infection Using a Whole-Genome RNA Interference Screen , 2017, mSphere.

[26]  N. Ridgway,et al.  Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System. , 2017, Cell reports.

[27]  Yasunori Yamamoto,et al.  The number of the C-terminal transmembrane domains has the potency to specify subcellular localization of Sec22c. , 2017, Biochemical and biophysical research communications.

[28]  C. Behl,et al.  The RAB GTPase RAB18 modulates macroautophagy and proteostasis. , 2017, Biochemical and biophysical research communications.

[29]  P. De Camilli,et al.  Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion , 2017, Science.

[30]  Xiaomin Luo,et al.  COPI–TRAPPII activates Rab18 and regulates its lipid droplet association , 2017, The EMBO journal.

[31]  D. G. Osborne,et al.  Structural and mechanistic insights into regulation of the retromer coat by TBC1d5 , 2016, Nature Communications.

[32]  Y. Goshima,et al.  Comprehensive behavioral study and proteomic analyses of CRMP2‐deficient mice , 2016, Genes to cells : devoted to molecular & cellular mechanisms.

[33]  W. Jackson,et al.  TBC1D20 mediates autophagy as a key regulator of autophagosome maturation , 2016, Autophagy.

[34]  A. Gropman,et al.  A Placebo-Controlled Trial of Simvastatin Therapy in Smith-Lemli-Opitz Syndrome , 2016, Genetics in Medicine.

[35]  T. Nilsson,et al.  The TRAPP Subunit Trs130p Interacts with the GAP Gyp6p to Mediate Ypt6p Dynamics at the Late Golgi , 2016, Front. Cell Dev. Biol..

[36]  Kenneth H. Roux,et al.  An improved smaller biotin ligase for BioID proximity labeling , 2016, Molecular biology of the cell.

[37]  Michael Davey,et al.  The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting , 2016, Molecular biology of the cell.

[38]  J. Briscoe,et al.  Reduced cholesterol levels impair Smoothened activation in Smith–Lemli–Opitz syndrome , 2015, Human molecular genetics.

[39]  Harald Stenmark,et al.  Cellular functions of Rab GTPases at a glance , 2015, Journal of Cell Science.

[40]  Mark T. Handley,et al.  Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation , 2015, Open Biology.

[41]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[42]  Darawalee Wangsa,et al.  Chromosome mis-segregation and cytokinesis failure in trisomic human cells , 2015, eLife.

[43]  C. Bucci,et al.  Repeated ER–endosome contacts promote endosome translocation and neurite outgrowth , 2015, Nature.

[44]  Kai Zhang,et al.  The structure of the dynactin complex and its interaction with dynein , 2015, Science.

[45]  C. Behrends,et al.  RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy , 2014, Autophagy.

[46]  S. Munro,et al.  Toward a Comprehensive Map of the Effectors of Rab GTPases , 2014, Developmental cell.

[47]  B. Bembi,et al.  Disorders of cholesterol metabolism and their unanticipated convergent mechanisms of disease. , 2014, Annual review of genomics and human genetics.

[48]  Marco Y. Hein,et al.  Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ * , 2014, Molecular & Cellular Proteomics.

[49]  M. Tagaya,et al.  Moonlighting functions of the NRZ (mammalian Dsl1) complex , 2014, Front. Cell Dev. Biol..

[50]  Mark T. Handley,et al.  Rab18 and a Rab18 GEF complex are required for normal ER structure , 2014, The Journal of cell biology.

[51]  Mark T. Handley,et al.  A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton , 2014, Disease Models & Mechanisms.

[52]  X. Darzacq,et al.  The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion , 2014, Nature Cell Biology.

[53]  Melissa C. Hendershott,et al.  Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin , 2014, Proceedings of the National Academy of Sciences.

[54]  K. Mirnics,et al.  Antioxidant Supplementation Ameliorates Molecular Deficits in Smith-Lemli-Opitz Syndrome , 2014, Biological Psychiatry.

[55]  Mark T. Handley,et al.  Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile mice and Warburg micro syndrome in humans. , 2013, American journal of human genetics.

[56]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[57]  J. Yates,et al.  REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture. , 2013, Developmental cell.

[58]  P. Wong,et al.  Comprehensive Gene Expression Profiling Reveals Synergistic Functional Networks in Cerebral Vessels after Hypertension or Hypercholesterolemia , 2013, PloS one.

[59]  R. Schekman,et al.  The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis , 2013, eLife.

[60]  M. Nowaczyk,et al.  Smith-Lemli-Opitz Syndrome , 2013 .

[61]  R. Steiner,et al.  Treatment of Smith–Lemli–Opitz syndrome and other sterol disorders , 2012, American journal of medical genetics. Part C, Seminars in medical genetics.

[62]  T. Pawson,et al.  Soluble FLT1 Binds Lipid Microdomains in Podocytes to Control Cell Morphology and Glomerular Barrier Function , 2012, Cell.

[63]  A. Spang.  The DSL1 Complex: The Smallest but Not the Least CATCHR , 2012, Traffic.

[64]  M. Tinti,et al.  Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates , 2012, Open Biology.

[65]  Brian Burke,et al.  A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells , 2012, The Journal of cell biology.

[66]  G. Frühbeck,et al.  Rab18 Dynamics in Adipocytes in Relation to Lipogenesis, Lipolysis and Obesity , 2011, PloS one.

[67]  R. Lothe,et al.  SPG20, a novel biomarker for early detection of colorectal cancer, encodes a regulator of cytokinesis , 2011, Oncogene.

[68]  Mark T. Handley,et al.  Loss-of-function mutations in RAB18 cause Warburg micro syndrome. , 2011, American journal of human genetics.

[69]  W. Wickner.  Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. , 2010, Annual review of cell and developmental biology.

[70]  D. Lambright,et al.  Rab GEFs and GAPs. , 2010, Current opinion in cell biology.

[71]  John D. Scott,et al.  Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties* , 2010, The Journal of Biological Chemistry.

[72]  T. Walz,et al.  A Structure-Based Mechanism for Vesicle Capture by the Multisubunit Tethering Complex Dsl1 , 2009, Cell.

[73]  N. Bright,et al.  Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5 , 2009, Journal of Cell Science.

[74]  W. Zwart,et al.  Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning , 2009, The Journal of cell biology.

[75]  P. Bieniasz,et al.  A role for ubiquitin ligases and Spartin/SPG20 in lipid droplet turnover , 2009, The Journal of cell biology.

[76]  R. Pepperkok,et al.  Rab18 and Rab43 have key roles in ER-Golgi trafficking , 2008, Journal of Cell Science.

[77]  M. Fukuda,et al.  Large Scale Screening for Novel Rab Effectors Reveals Unexpected Broad Rab Binding Specificity*S , 2008, Molecular & Cellular Proteomics.

[78]  M. Lowe,et al.  Targeting of the type II inositol polyphosphate 5-phosphatase INPP5B to the early secretory pathway , 2007, Journal of Cell Science.

[79]  G. Corso,et al.  Clinical phenotype of lathosterolosis , 2007, American journal of medical genetics. Part A.

[80]  M. Malagón,et al.  Rab18 Inhibits Secretory Activity in Neuroendocrine Cells by Interacting with Secretory Granules , 2007, Traffic.

[81]  H. Cai,et al.  Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. , 2007, Developmental cell.

[82]  A. Grinberg,et al.  Development and characterization of a hypomorphic Smith-Lemli-Opitz syndrome mouse model and efficacy of simvastatin therapy. , 2006, Human molecular genetics.

[83]  M. Zerial,et al.  Regulated Localization of Rab18 to Lipid Droplets , 2005, Journal of Biological Chemistry.

[84]  T. Cover,et al.  The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments. , 2005, Molecular biology of the cell.

[85]  James H. Hurley,et al.  Structural mechanism for sterol sensing and transport by OSBP-related proteins , 2005, Nature.

[86]  H. Taniguchi,et al.  Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane , 2005, Journal of Cell Science.

[87]  Colin A. Johnson,et al.  Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome , 2005, Nature Genetics.

[88]  M. D'Esposito,et al.  Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators. , 2004, Trends in biochemical sciences.

[89]  A. Hunter,et al.  Lathosterolosis: an inborn error of human and murine cholesterol synthesis due to lathosterol 5-desaturase deficiency. , 2003, Human molecular genetics.

[90]  A. Ballabio,et al.  Lathosterolosis, a Novel Multiple-Malformation/Mental Retardation Syndrome Due to Deficiency of 3β-Hydroxysteroid-Δ5-Desaturase , 2002 .

[91]  T. Südhof,et al.  Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif. , 2002, Developmental cell.

[92]  F. RivasCrespo,et al.  [Smith-Lemli-Opitz syndrome]. , 2001, Anales espanoles de pediatria.

[93]  H. McBride,et al.  The Rab5 effector EEA1 is a core component of endosome docking , 1999, Nature.

[94]  S. Wong,et al.  Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment. , 1999, Molecular biology of the cell.

[95]  R. Wevers,et al.  New treatment strategy for Smith-Lemli-Opitz syndrome , 1997, The Lancet.

[96]  M. Kaghad,et al.  Emopamil-binding Protein, a Mammalian Protein That Binds a Series of Structurally Diverse Neuroprotective Agents, Exhibits Δ8-Δ7 Sterol Isomerase Activity in Yeast* , 1996, The Journal of Biological Chemistry.

[97]  K. Oughstun.  Evolution of the Signal , 2019, Springer Series in Optical Sciences.

[98]  R. Parton,et al.  ORP2 Delivers Cholesterol to the Plasma Membrane in Exchange for Phosphatidylinositol 4, 5-Bisphosphate (PI(4,5)P2). , 2019, Molecular cell.

[99]  S. Tam,et al.  Lathosterolosis: a disorder of cholesterol biosynthesis resembling smith-lemli-opitz syndrome. , 2014, JIMD reports.

[100]  B. Wollnik,et al.  A homozygous RAB3GAP2 mutation causes Warburg Micro syndrome , 2010, Human Genetics.

[101]  M. Fukuda,et al.  Large-scale Screening for Novel Rab Effectors Reveals Unexpected Broad Rab-binding Specificity* , 2008 .

[102]  B. Horazdovsky,et al.  Vps9 domain-containing proteins: activators of Rab5 GTPases from yeast to neurons. , 2006, Trends in cell biology.

[103]  A. Ballabio,et al.  Lathosterolosis, a novel multiple-malformation/mental retardation syndrome due to deficiency of 3beta-hydroxysteroid-delta5-desaturase. , 2002, American journal of human genetics.