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PMID: 15078904 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo.

The Journal of cell biology ·Vol. 165 ·No. 1 ·2004-04-00 ·Pages 135-44

Dunn R, Klos DA, Adler AS, Hicke L

Abstract

Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently. The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells. Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo. Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting. These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides.

MeSH Terms
Carrier Proteins/metabolism Endosomal Sorting Complexes Required for Transport Endosomes/metabolism,ultrastructure Intracellular Membranes/metabolism,ultrastructure Lysine/metabolism Membrane Proteins/metabolism Mutation/genetics Phosphatidylinositols/metabolism Protein Binding/genetics Protein Structure, Tertiary/genetics Protein Transport/genetics Saccharomyces cerevisiae/enzymology,genetics,ultrastructure Saccharomyces cerevisiae Proteins/metabolism Transport Vesicles/metabolism,ultrastructure Ubiquitin/metabolism Ubiquitin-Protein Ligase Complexes/metabolism
Chemicals
Carrier Proteins Endosomal Sorting Complexes Required for Transport Membrane Proteins Phosphatidylinositols Saccharomyces cerevisiae Proteins Ubiquitin Ubiquitin-Protein Ligase Complexes RSP5 protein, S cerevisiae Lysine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Dunn Rebecca
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Hogan 2-100, 2205 Tech Dr., Evanston, IL 60208-3500, USA.
Klos Deborah A
Adler Adam S
Hicke Linda
References (55)
55 references, click to expand
  1. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association.
    Cell. 1999 Oct 29;99(3):323-34 PMID: 10555148
  2. Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae.
    Mol Cell Biol. 1999 Oct;19(10):6972-9 PMID: 10490634
  3. High-yield expression and purification of recombinant proteins in bacteria: a versatile vector for glutathione S-transferase fusion proteins containing two protease cleavage sites.
    Anal Biochem. 2000 Jun 1;281(2):232-4 PMID: 10870841
  4. Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases.
    J Membr Biol. 2000 Jul 1;176(1):1-17 PMID: 10882424
  5. Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II.
    J Biol Chem. 2000 Jul 7;275(27):20562-71 PMID: 10781604
  6. Binding kinetics and ligand specificity for the interactions of the C2B domain of synaptogmin II with inositol polyphosphates and phosphoinositides.
    Biochemistry. 2000 Aug 15;39(32):9679-86 PMID: 10933784
  7. Signaling and subcellular targeting by membrane-binding domains.
    Annu Rev Biophys Biomol Struct. 2000;29:49-79 PMID: 10940243
  8. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.
    Cell. 2000 Sep 1;102(5):577-86 PMID: 11007476
  9. Basic Medical Research Award. The ubiquitin system.
    Nat Med. 2000 Oct;6(10):1073-81 PMID: 11017125
  10. Polar transmembrane domains target proteins to the interior of the yeast vacuole.
    Mol Biol Cell. 2000 Nov;11(11):3737-49 PMID: 11071903
  11. Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis.
    Mol Biol Cell. 2001 Feb;12(2):421-35 PMID: 11179425
  12. Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.
    Science. 2001 Feb 9;291(5506):1047-51 PMID: 11161217
  13. Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking.
    Traffic. 2000 Mar;1(3):248-58 PMID: 11208108
  14. TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes.
    J Biol Chem. 2001 Apr 13;276(15):11735-42 PMID: 11134028
  15. Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway.
    Mol Cell Biol. 2001 May;21(10):3564-75 PMID: 11313482
  16. Inositol lipids as spatial regulators of membrane traffic.
    J Membr Biol. 2001 Apr 1;180(3):187-94 PMID: 11337890
  17. Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease.
    J Cell Biol. 2001 May 14;153(4):649-62 PMID: 11352928
  18. Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase.
    Mol Cell Biol. 2001 Jul;21(14):4482-94 PMID: 11416128
  19. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I.
    Cell. 2001 Jul 27;106(2):145-55 PMID: 11511343
  20. Membrane targeting by C1 and C2 domains.
    J Biol Chem. 2001 Aug 31;276(35):32407-10 PMID: 11432875
  21. Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole.
    Traffic. 2001 Sep;2(9):622-30 PMID: 11555416
  22. Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting.
    EMBO J. 2001 Sep 17;20(18):5176-86 PMID: 11566881
  23. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.
    Cell. 2001 Oct 5;107(1):55-65 PMID: 11595185
  24. Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1.
    J Biol Chem. 2001 Nov 23;276(47):43949-57 PMID: 11500494
  25. Human VPS34 is required for internal vesicle formation within multivesicular endosomes.
    J Cell Biol. 2001 Dec 24;155(7):1251-64 PMID: 11756475
  26. Mechanisms underlying ubiquitination.
    Annu Rev Biochem. 2001;70:503-33 PMID: 11395416
  27. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies.
    Nat Cell Biol. 2002 Feb;4(2):117-23 PMID: 11788821
  28. A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins.
    Nature. 2002 Mar 28;416(6879):451-5 PMID: 11919637
  29. Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis.
    Nat Cell Biol. 2002 May;4(5):389-93 PMID: 11988742
  30. Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body.
    Curr Biol. 2002 Jun 4;12(11):885-93 PMID: 12062051
  31. Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor.
    Neuron. 2002 Aug 1;35(3):447-59 PMID: 12165468
  32. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting.
    Dev Cell. 2002 Aug;3(2):271-82 PMID: 12194857
  33. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body.
    Dev Cell. 2002 Aug;3(2):283-9 PMID: 12194858
  34. Comm sorts robo to control axon guidance at the Drosophila midline.
    Cell. 2002 Aug 23;110(4):415-27 PMID: 12202032
  35. The C2A domain of JFC1 binds to 3'-phosphorylated phosphoinositides and directs plasma membrane association in living cells.
    Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11652-7 PMID: 12189202
  36. Receptor downregulation and multivesicular-body sorting.
    Nat Rev Mol Cell Biol. 2002 Dec;3(12):893-905 PMID: 12461556
  37. C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling.
    Biochemistry. 2003 Feb 11;42(5):1254-65 PMID: 12564928
  38. Protein sorting into multivesicular endosomes.
    Curr Opin Cell Biol. 2003 Aug;15(4):446-55 PMID: 12892785
  39. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.
    Annu Rev Cell Dev Biol. 2003;19:141-72 PMID: 14570567
  40. Getting started with yeast.
    Methods Enzymol. 1991;194:3-21 PMID: 2005794
  41. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.
    Mol Biol Cell. 1992 Dec;3(12):1389-402 PMID: 1493335
  42. Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.
    J Cell Biol. 1993 Feb;120(4):865-75 PMID: 8432727
  43. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II.
    J Biol Chem. 1994 Nov 18;269(46):29206-11 PMID: 7961887
  44. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.
    J Cell Biol. 1995 Mar;128(5):779-92 PMID: 7533169
  45. Rapid protein extraction from Saccharomyces cerevisiae.
    Yeast. 1994 Oct;10(10):1305-10 PMID: 7900419
  46. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis.
    Cell. 1996 Jan 26;84(2):277-87 PMID: 8565073
  47. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease.
    J Biol Chem. 1996 May 3;271(18):10946-52 PMID: 8631913
  48. Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin.
    Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13327-32 PMID: 8917590
  49. The C2 domain calcium-binding motif: structural and functional diversity.
    Protein Sci. 1996 Dec;5(12):2375-90 PMID: 8976547
  50. The C2 domain of the ubiquitin protein ligase Nedd4 mediates Ca2+-dependent plasma membrane localization.
    J Biol Chem. 1997 Dec 19;272(51):32329-36 PMID: 9405440
  51. The C2 domains of Rabphilin3A specifically bind phosphatidylinositol 4,5-bisphosphate containing vesicles in a Ca2+-dependent manner. In vitro characteristics and possible significance.
    J Biol Chem. 1998 Apr 24;273(17):10240-8 PMID: 9553075
  52. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function.
    EMBO J. 1998 Jun 1;17(11):2982-93 PMID: 9606181
  53. Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains.
    J Biol Chem. 1998 Nov 13;273(46):30497-508 PMID: 9804818
  54. Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body.
    Cell. 1998 Dec 11;95(6):847-58 PMID: 9865702
  55. Phosphoinositide signaling and the regulation of membrane trafficking in yeast.
    Trends Biochem Sci. 2000 May;25(5):229-35 PMID: 10782093
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.200309026
Published
2004-04-00
Pages
135-44
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2172079
Subset
IM
Grants
NIDDK NIH HHS · R01 DK061299 · United States
NIDDK NIH HHS · R56 DK061299 · United States
NIDDK NIH HHS · DK61299 · United States
External Links
PubMed source DOI: 10.1083/jcb.200309026 PMC Full Text View journal details
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