Comparative proximity biotinylation implicates RAB18 in sterol mobilization and biosynthesis

ABSTRACT Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation in HEK293 and HeLa cells to generate an inventory of potential RAB18 effectors. In HeLa cells, we expressed BirA*-RAB18 in cells in which RAB18-guanine nucleotide exchange factor (GEF) activity was disrupted with CRISPR. We found that most RAB18-interactions are regulated independently by its different GEFs; the binary RAB3GAP1-RAB3GAP2 complex and the TRAPPC9-containing TRAPPII complex. RAB3GAP-dependent RAB18 interactions included a group of microtubule-interacting/membrane shaping proteins, a group of proteins involved in membrane tethering and docking, and a group of lipid-modifying/lipid transport proteins. We demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation. Further, we provide confirmatory evidence for several of the interactors (SPG20/SPART, SEC22A and TMCO4) as well as functional evidence supporting a role for RAB18 in modulating the close apposition of membranes and in cholesterol mobilization. SUMMARY STATEMENT We used proximity biotinylation together with guanine nucleotide exchange factor (GEF)-null cell lines to discriminate functional RAB18-interactions. We anticipate that this approach will be broadly applicable in small GTPase research.

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

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

[3]  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.

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

[5]  Max A. Horlbeck,et al.  New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells , 2019, bioRxiv.

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

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

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

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

[10]  S. Eimer,et al.  RAB18 impacts autophagy via lipid droplet-derived lipid transfer and is rescued by ATG9A , 2018, bioRxiv.

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

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

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

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

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

[16]  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.

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

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

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

[20]  C. Abraham,et al.  Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin , 2017, Microbial cell.

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

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

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

[24]  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.

[25]  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.

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

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

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

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

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

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

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

[33]  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..

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

[35]  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.

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

[37]  J. Simpson,et al.  A high-content screening microscopy approach to dissect the role of Rab proteins in Golgi-to-ER retrograde trafficking , 2015, Journal of Cell Science.

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

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

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

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

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

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

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

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

[46]  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.

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

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

[49]  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.

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

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

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

[53]  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.

[54]  B. Burke,et al.  BioID: A Screen for Protein‐Protein Interactions , 2013, Current protocols in protein science.

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

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

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

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

[59]  Seungbok Lee,et al.  Spartin Regulates Synaptic Growth and Neuronal Survival by Inhibiting BMP-Mediated Microtubule Stabilization , 2013, Neuron.

[60]  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.

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

[62]  C. Blackstone,et al.  Spg20-/- mice reveal multimodal functions for Troyer syndrome protein spartin in lipid droplet maintenance, cytokinesis and BMP signaling. , 2012, Human molecular genetics.

[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]  J. C. Bakowska,et al.  SPG20 Protein Spartin Associates with Cardiolipin via Its Plant-Related Senescence Domain and Regulates Mitochondrial Ca2+ Homeostasis , 2011, PloS one.

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

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

[70]  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.

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

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

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

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

[75]  Xinnan Wang,et al.  The hereditary spastic paraplegia proteins NIPA1, spastin and spartin are inhibitors of mammalian BMP signalling , 2009, Human molecular genetics.

[76]  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.

[77]  J. Luzio,et al.  Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5 , 2009, The Biochemical journal.

[78]  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.

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

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

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

[82]  H. Schwarz,et al.  Ypt1p is essential for retrograde Golgi-ER transport and for Golgi maintenance in S. cerevisiae , 2008, Journal of Cell Science.

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

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

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

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

[87]  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.

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

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

[90]  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.

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

[92]  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.

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

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

[95]  M. Tagaya,et al.  Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion , 2004, The EMBO journal.

[96]  M. Tohyama,et al.  Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi , 2004, The EMBO journal.

[97]  E. Hartmann,et al.  Use1p is a yeast SNARE protein required for retrograde traffic to the ER , 2003, The EMBO journal.

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

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

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

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

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

[103]  J. Rayner,et al.  A novel SNARE complex implicated in vesicle fusion with the endoplasmic reticulum , 1997, The EMBO journal.

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

[105]  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.

[106]  H. Pelham,et al.  SNARE-Mediated Retrograde Traffic from the Golgi Complex to the Endoplasmic Reticulum , 1996, Cell.