The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit.

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

[1]  J. Bonifacino,et al.  PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal. , 2007, Molecular biology of the cell.

[2]  R. Schekman,et al.  Exomer: a coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast , 2006, The Journal of cell biology.

[3]  G. E. Fernandez,et al.  Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p. , 2006, Molecular biology of the cell.

[4]  J. Hurley,et al.  Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein–related proteins and phosphoinositides , 2006, The Journal of cell biology.

[5]  Christina Schindler,et al.  Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi , 2006, The EMBO journal.

[6]  C. Czupalla,et al.  Proteomic analysis of adaptor protein 1A coats selectively assembled on liposomes. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Verkade,et al.  Caveolin-1 Is Not Essential for Biosynthetic Apical Membrane Transport , 2005, Molecular and Cellular Biology.

[8]  P. Cullen,et al.  Coincidence detection in phosphoinositide signaling. , 2005, Trends in cell biology.

[9]  T. Stevens,et al.  Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae. , 2005, Biochimica et biophysica acta.

[10]  A. Godi,et al.  The role of the phosphoinositides at the Golgi complex. , 2005, Biochimica et biophysica acta.

[11]  Michael Krauss,et al.  Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. , 2005, Molecular cell.

[12]  T. Balla.  Inositol-lipid binding motifs: signal integrators through protein-lipid and protein-protein interactions , 2005, Journal of Cell Science.

[13]  A. Parsons,et al.  Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway. , 2004, Molecular biology of the cell.

[14]  T. Levine,et al.  Multiple Pools of Phosphatidylinositol 4-Phosphate Detected Using the Pleckstrin Homology Domain of Osh2p* , 2004, Journal of Biological Chemistry.

[15]  S. Harrison,et al.  Crystal structure of the clathrin adaptor protein 1 core. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Masahiro Shibata,et al.  Insights into the Phosphoregulation of β‐Secretase Sorting Signal by the VHS Domain of GGA1 , 2004, Traffic.

[17]  M. Robinson.  Adaptable adaptors for coated vesicles. , 2004, Trends in cell biology.

[18]  Diana Murray,et al.  Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. , 2004, Molecular cell.

[19]  A. D. Robertson,et al.  GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network , 2004, Nature Cell Biology.

[20]  P. McPherson,et al.  ENTH/ANTH proteins and clathrin-mediated membrane budding , 2004, Journal of Cell Science.

[21]  Rodrigo Lopez,et al.  Multiple sequence alignment with the Clustal series of programs , 2003, Nucleic Acids Res..

[22]  M. Lemmon,et al.  Phosphoinositide Recognition Domains , 2003, Traffic.

[23]  Ian G. Mills,et al.  Curvature of clathrin-coated pits driven by epsin , 2002, Nature.

[24]  P. Salo,et al.  ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization. , 2002, Molecular biology of the cell.

[25]  K. Nakayama,et al.  GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors. , 2002, The Biochemical journal.

[26]  P. Evans,et al.  Molecular Architecture and Functional Model of the Endocytic AP2 Complex , 2002, Cell.

[27]  S. Munro,et al.  Targeting of Golgi-Specific Pleckstrin Homology Domains Involves Both PtdIns 4-Kinase-Dependent and -Independent Components , 2002, Current Biology.

[28]  G. Prestwich,et al.  Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex , 2002, The Journal of cell biology.

[29]  R. Schekman,et al.  The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins. , 2002, Developmental cell.

[30]  Alex Bateman,et al.  The ENTH domain , 2002, FEBS letters.

[31]  J. Gerst,et al.  Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast , 2002, The EMBO journal.

[32]  R. Schekman,et al.  A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway , 2002, The Journal of cell biology.

[33]  Gary D Bader,et al.  Systematic Genetic Analysis with Ordered Arrays of Yeast Deletion Mutants , 2001, Science.

[34]  A. Boman.  GGA proteins: new players in the sorting game. , 2001, Journal of cell science.

[35]  S. Emr,et al.  Yeast Gga coat proteins function with clathrin in Golgi to endosome transport. , 2001, Molecular biology of the cell.

[36]  S. Munro,et al.  Dual targeting of Osh1p, a yeast homologue of oxysterol-binding protein, to both the Golgi and the nucleus-vacuole junction. , 2001, Molecular biology of the cell.

[37]  P. Novick,et al.  Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p. , 2001, Molecular biology of the cell.

[38]  P R Evans,et al.  Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. , 2001, Science.

[39]  T. Kigawa,et al.  Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. , 2001, Science.

[40]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[41]  O. Zhdankina,et al.  Yeast GGA proteins interact with GTP‐bound Arf and facilitate transport through the Golgi , 2001, Yeast.

[42]  H. Pelham,et al.  A Selective Transport Route from Golgi to Late Endosomes That Requires the Yeast Gga Proteins , 2000, The Journal of cell biology.

[43]  M. Foti,et al.  Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics. , 2000, Molecular biology of the cell.

[44]  M. Sternberg,et al.  Enhanced genome annotation using structural profiles in the program 3D-PSSM. , 2000, Journal of molecular biology.

[45]  P. Novick,et al.  The yeast phosphatidylinositol-4-OH kinase Pik1 regulates secretion at the Golgi , 1999, Nature Cell Biology.

[46]  J. Thorner,et al.  Direct Involvement of Phosphatidylinositol 4-Phosphate in Secretion in the Yeast Saccharomyces cerevisiae * , 1999, The Journal of Biological Chemistry.

[47]  A. Luini,et al.  ARF mediates recruitment of PtdIns-4-OH kinase-β and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex , 1999, Nature Cell Biology.

[48]  I. Gaidarov,et al.  Phosphoinositide–Ap-2 Interactions Required for Targeting to Plasma Membrane Clathrin-Coated Pits , 1999, The Journal of cell biology.

[49]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[50]  C Sander,et al.  Mapping the Protein Universe , 1996, Science.

[51]  P. Novick,et al.  The role of Myo2, a yeast class V myosin, in vesicular transport , 1995, The Journal of cell biology.

[52]  K. Takegawa,et al.  Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. , 1993, Science.

[53]  P. Novick,et al.  Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain , 1990, The Journal of cell biology.

[54]  R. Schiestl,et al.  High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier , 1989, Current Genetics.

[55]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[56]  R. Schekman,et al.  Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway , 1980, Cell.

[57]  J. Bonifacino.  The GGA proteins: adaptors on the move , 2004, Nature Reviews Molecular Cell Biology.

[58]  S. Kornfeld,et al.  The GGA proteins: key players in protein sorting at the trans-Golgi network. , 2004, European journal of cell biology.

[59]  T. Müller-Reichert,et al.  Cryomethods for thin section electron microscopy. , 2002, Methods in enzymology.

[60]  H. Pelham,et al.  Two syntaxin homologues in the TGN/endosomal system of yeast , 1998, The EMBO journal.

[61]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .