User menu

Accès à distance ? S'identifier sur le proxy UCLouvain

Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway.

Primary tabs

download
  • Open access
  • PDF
  • 763.08 K
Xia, Zanxian Webster, Ailsa Du, Fangyong Piatkov, Konstantin Ghislain, Michel [UCL] Varshavsky, Alexander

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first approximately 700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated approximately 70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a K(d) of approximately 1 microm, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

Document type Article de périodique (Journal article) – Article de recherche – Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
Access type Accès libre
Publication date 2008
Language Anglais
Journal information "The Journal of biological chemistry" - Vol. 283, no. 35, p. 24011-28 (2008)
Peer reviewed yes
issn 0021-9258
Publication status Publié
Affiliation UCL - AGRO/CABI - Département de chimie appliquée et des bio-industries
MESH Subject Amino Acid Motifs - physiology ; Binding Sites - physiology ; Dipeptides - genetics - metabolism ; Homeodomain Proteins - genetics - metabolism ; Mutation ; Peptide Mapping - methods ; Protein Binding ; Saccharomyces cerevisiae - enzymology - genetics ; Saccharomyces cerevisiae Proteins - genetics - metabolism ; Transcription Factors - genetics - metabolism ; Ubiquitin - genetics - metabolism ; Ubiquitin-Protein Ligases - genetics - metabolism ; Ubiquitination - physiology
Links
  1. Varshavsky A., The N-end rule: functions, mysteries, uses., 10.1073/pnas.93.22.12142
  2. Tobias J., Shrader T., Rocap G, Varshavsky A, The N-end rule in bacteria, 10.1126/science.1962196
  3. Shrader T E, Tobias J W, Varshavsky A, The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat., 10.1128/jb.175.14.4364-4374.1993
  4. Graciet E., Hu R.-G., Piatkov K., Rhee J. H., Schwarz E. M., Varshavsky A., Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen, 10.1073/pnas.0511224103
  5. Erbse A., Schmidt R., Bornemann T., Schneider-Mergener J., Mogk A., Zahn R., Dougan D. A., Bukau B., ClpS is an essential component of the N-end rule pathway in Escherichia coli, 10.1038/nature04412
  6. Genes Dev., 15, 403 (2007)
  7. Hou Jennifer Y, Sauer Robert T, Baker Tania A, Distinct structural elements of the adaptor ClpS are required for regulating degradation by ClpAP, 10.1038/nsmb.1392
  8. Suto Kyoko, Shimizu Yoshihiro, Watanabe Kazunori, Ueda Takuya, Fukai Shuya, Nureki Osamu, Tomita Kozo, Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog, 10.1038/sj.emboj.7601433
  9. Watanabe Kazunori, Toh Yukimatsu, Suto Kyoko, Shimizu Yoshihiro, Oka Natsuhisa, Wada Takeshi, Tomita Kozo, Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase, 10.1038/nature06167
  10. Dong X., Kato-Murayama M., Muramatsu T., Mori H., Shirouzu M., Bessho Y., Yokoyama S., The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli, 10.1110/ps.062616107
  11. Bachmair Andreas, Varshavsky Alexander, The degradation signal in a short-lived protein, 10.1016/0092-8674(89)90635-1
  12. Mogk Axel, Schmidt Ronny, Bukau Bernd, The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies, 10.1016/j.tcb.2007.02.001
  13. Johnson Erica S., Gonda David K., Varshavsky Alexander, Cis-trans recognition and subunit-specific degradation of short-lived proteins, 10.1038/346287a0
  14. Suzuki T., Degradation signals in the lysine-asparagine sequence space, 10.1093/emboj/18.21.6017
  15. Prakash Sumit, Tian Lin, Ratliff Kevin S, Lehotzky Rebecca E, Matouschek Andreas, An unstructured initiation site is required for efficient proteasome-mediated degradation, 10.1038/nsmb814
  16. Inobe Tomonao, Matouschek Andreas, Protein targeting to ATP-dependent proteases, 10.1016/j.sbi.2007.12.014
  17. Baker Rohan T., Varshavsky Alexander, Yeast N-terminal Amidase : A NEW ENZYME AND COMPONENT OF THE N-END RULE PATHWAY, 10.1074/jbc.270.20.12065
  18. Kwon Y. T., Balogh S. A., Davydov I. V., Kashina A. S., Yoon J. K., Xie Y., Gaur A., Hyde L., Denenberg V. H., Varshavsky A., Altered Activity, Social Behavior, and Spatial Memory in Mice Lacking the NTAN1p Amidase and the Asparagine Branch of the N-End Rule Pathway, 10.1128/mcb.20.11.4135-4148.2000
  19. Kwon Yong Tae, Kashina Anna S., Varshavsky Alexander, Alternative Splicing Results in Differential Expression, Activity, and Localization of the Two Forms of Arginyl-tRNA-Protein Transferase, a Component of the N-End Rule Pathway, 10.1128/mcb.19.1.182
  20. Kwon Y. T., An Essential Role of N-Terminal Arginylation in Cardiovascular Development, 10.1126/science.1069531
  21. J. Biol. Chem., 264, 16700 (1989)
  22. Tasaki Takafumi, Sohr Reinhard, Xia Zanxian, Hellweg Rainer, Hörtnagl Heide, Varshavsky Alexander, Kwon Yong Tae, Biochemical and Genetic Studies of UBR3, a Ubiquitin Ligase with a Function in Olfactory and Other Sensory Systems, 10.1074/jbc.m701894200
  23. Tasaki Takafumi, Kwon Yong Tae, The mammalian N-end rule pathway: new insights into its components and physiological roles, 10.1016/j.tibs.2007.08.010
  24. Kwon Y. T., Xia Z., Davydov I. V., Lecker S. H., Varshavsky A., Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3alpha ) of the N-End Rule Pathway, 10.1128/mcb.21.23.8007-8021.2001
  25. Kwon Yong Tae, Xia Zanxian, An Jee Young, Tasaki Takafumi, Davydov Ilia V., Seo Jai Wha, Sheng Jun, Xie Youming, Varshavsky Alexander, Female Lethality and Apoptosis of Spermatocytes in Mice Lacking the UBR2 Ubiquitin Ligase of the N-End Rule Pathway, 10.1128/mcb.23.22.8255-8271.2003
  26. Tasaki T., Mulder L. C. F., Iwamatsu A., Lee M. J., Davydov I. V., Varshavsky A., Muesing M., Kwon Y. T., A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons, 10.1128/mcb.25.16.7120-7136.2005
  27. An J. Y., Seo J. W., Tasaki T., Lee M. J., Varshavsky A., Kwon Y. T., Impaired neurogenesis and cardiovascular development in mice lacking the E3 ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway, 10.1073/pnas.0601700103
  28. Stary S., PRT1 of Arabidopsis Is a Ubiquitin Protein Ligase of the Plant N-End Rule Pathway with Specificity for Aromatic Amino-Terminal Residues, 10.1104/pp.103.029272
  29. Garzón Marcus, Eifler Karolin, Faust Andrea, Scheel Hartmut, Hofmann Kay, Koncz Csaba, Yephremov Alexander, Bachmair Andreas, PRT6/At5g02310 encodes anArabidopsisubiquitin ligase of the N-end rule pathway with arginine specificity and is not theCER3locus, 10.1016/j.febslet.2007.06.005
  30. Siepmann Thomas J., Bohnsack Richard N., Tokgöz Zeynep, Baboshina Olga V., Haas Arthur L., Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway, 10.1074/jbc.m211240200
  31. Kwak Keith S., Zhou Xiaolan, Solomon Vered, Baracos Vickie E., Davis James, Bannon Anthony W., Boyle William J., Lacey David L., Han H.Q., Regulation of Protein Catabolism by Muscle-Specific and Cytokine-Inducible Ubiquitin Ligase E3α-II during Cancer Cachexia, 10.1158/0008-5472.can-04-2102
  32. Varshavsky Alexander, ‘Spalog’ and ‘sequelog’: neutral terms for spatial and sequence similarity, 10.1016/j.cub.2004.02.014
  33. Byrd C., The N-end rule pathway controls the import of peptides through degradation of a transcriptional repressor, 10.1093/emboj/17.1.269
  34. Hu R.-G., Wang H., Xia Z., Varshavsky A., The N-end rule pathway is a sensor of heme, 10.1073/pnas.0710568105
  35. Varshavsky Alexander, Turner Glenn C., Du Fangyong, 10.1038/35014629
  36. Alagramam Kumar, Naider Fred, Becker Jeffrey M., A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae, 10.1111/j.1365-2958.1995.tb02237.x
  37. Lupas Andrei N, Koretke Kristin K, Bioinformatic analysis of ClpS, a protein module involved in prokaryotic and eukaryotic protein degradation, 10.1016/s1047-8477(02)00582-8
  38. Rao Hai, Uhlmann Frank, Nasmyth Kim, Varshavsky Alexander, Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability, 10.1038/35073627
  39. Ditzel Mark, Wilson Rebecca, Tenev Tencho, Zachariou Anna, Paul Angela, Deas Emma, Meier Pascal, Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis, 10.1038/ncb984
  40. Varshavsky Alexander, The N-end rule and regulation of apoptosis, 10.1038/ncb0503-373
  41. Sasaki Takanori, Kojima Hirotada, Kishimoto Rikiya, Ikeda Ayu, Kunimoto Hiroyuki, Nakajima Koich, Spatiotemporal Regulation of c-Fos by ERK5 and the E3 Ubiquitin Ligase UBR1, and Its Biological Role, 10.1016/j.molcel.2006.08.005
  42. Lloyd Aliza G., Ng Yen Shing, Muesing Mark A., Simon Viviana, Mulder Lubbertus C.F., Characterization of HIV-1 integrase N-terminal mutant viruses, 10.1016/j.virol.2006.10.007
  43. Yoshida Satoko, Ito Masaki, Callis Judy, Nishida Ikuo, Watanabe Akira, A delayed leaf senescence mutant is defective in arginyl-tRNA:protein arginyltransferase, a component of the N-end rule pathway inArabidopsis, 10.1046/j.1365-313x.2002.01407.x
  44. Zenker Martin, Mayerle Julia, Lerch Markus M, Tagariello Andreas, Zerres Klaus, Durie Peter R, Beier Matthias, Hülskamp Georg, Guzman Celina, Rehder Helga, Beemer Frits A, Hamel Ben, Vanlieferinghen Philippe, Gershoni-Baruch Ruth, Vieira Marta W, Dumic Miroslav, Auslender Ron, Gil-da-Silva-Lopes Vera L, Steinlicht Simone, Rauh Manfred, Shalev Stavit A, Thiel Christian, Winterpacht Andreas, Kwon Yong Tae, Varshavsky Alexander, Reis André, Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome), 10.1038/ng1681
  45. Hu Rong-Gui, Brower Christopher S., Wang Haiqing, Davydov Ilia V., Sheng Jun, Zhou Jianmin, Kwon Yong Tae, Varshavsky Alexander, Arginyltransferase, Its Specificity, Putative Substrates, Bidirectional Promoter, and Splicing-derived Isoforms, 10.1074/jbc.m604355200
  46. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl, K. (2006) Current Protocols in Molecular Biology, Wiley-Interscience, New York
  47. CHEN E, KUANG W, LEE A, Overview of manual and automated DNA sequencing by the dideoxy chain termination method, 10.1016/s1046-2023(05)80158-6
  48. EMBO J., 15, 4884 (1996)
  49. Barral Y, Jentsch S, Mann C, G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast., 10.1101/gad.9.4.399
  50. Bachmair A, Finley D, Varshavsky A, In vivo half-life of a protein is a function of its amino-terminal residue, 10.1126/science.3018930
  51. Varshavsky Alexander, Ubiquitin Fusion Technique and Related Methods, Methods in Enzymology (2005) ISBN:9780121828042 p.777-799, 10.1016/s0076-6879(05)99051-4
  52. Johnson Erica S., Ma Philip C. M., Ota Irene M., Varshavsky Alexander, A Proteolytic Pathway That Recognizes Ubiquitin as a Degradation Signal, 10.1074/jbc.270.29.17442
  53. Levy F., Johnsson N., Rumenapf T., Varshavsky A., Using ubiquitin to follow the metabolic fate of a protein., 10.1073/pnas.93.10.4907
  54. Genetics, 122, 19 (1989)
  55. Gietz R.Daniel, Akio Sugino, New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites, 10.1016/0378-1119(88)90185-0
  56. Hu Rong-Gui, Sheng Jun, Qi Xin, Xu Zhenming, Takahashi Terry T., Varshavsky Alexander, The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators, 10.1038/nature04027
  57. Del Tito B J, Ward J M, Hodgson J, Gershater C J, Edwards H, Wysocki L A, Watson F A, Sathe G, Kane J F, Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli., 10.1128/jb.177.24.7086-7091.1995
  58. Stevens Scott W, [26] Analysis of low-abundance ribonucleoprotein particles from yeast by affinity chromatography and mass spectrometry microsequencing, Methods in Enzymology (2000) ISBN:9780121822194 p.385-398, 10.1016/s0076-6879(00)18065-6
  59. de Groot R. J., Rumenapf T., Kuhn R. J., Strauss E. G., Strauss J. H., Sindbis virus RNA polymerase is degraded by the N-end rule pathway., 10.1073/pnas.88.20.8967
  60. Seeliger Markus A, Breward Sadie E, Friedler Assaf, Schon Oliver, Itzhaki Laura S, Cooperative organization in a macromolecular complex, 10.1038/nsb962
  61. Moore Jamie M. R., Galicia Sarah J., McReynolds Andrea C., Nguyen Ngoc-Ha, Scanlan Thomas S., Guy R. Kiplin, Quantitative Proteomics of the Thyroid Hormone Receptor-Coregulator Interactions, 10.1074/jbc.m403453200
  62. Myszka David G, Morton Thomas A, CLAMP©: a biosensor kinetic data analysis program, 10.1016/s0968-0004(98)01183-9
  63. Brock Susannah C., Bonsall Joanna, Luskin Marla B., The Neuronal Progenitor Cells of the Forebrain Subventricular Zone: Intrinsic Propertiesin Vitroand Following Transplantation, 10.1006/meth.1998.0684
  64. Du, F. (2001) Allosteric Activation of the Ubiquitin Ligase UBR1 by Short Peptides: Molecular Mechanisms and Physiological Functions. Ph.D. thesis, California Institute of Technology, Pasadena, CA
  65. Feldman R.M.Renny, Correll Craig C, Kaplan Kenneth B, Deshaies Raymond J, A Complex of Cdc4p, Skp1p, and Cdc53p/Cullin Catalyzes Ubiquitination of the Phosphorylated CDK Inhibitor Sic1p, 10.1016/s0092-8674(00)80404-3
  66. Banerji J., Sands J., Strominger J. L., Spies T., A gene pair from the human major histocompatibility complex encodes large proline-rich proteins with multiple repeated motifs and a single ubiquitin-like domain., 10.1073/pnas.87.6.2374
  67. EMBO J., 9, 3179 (1990)
  68. Lee M. J., Tasaki T., Moroi K., An J. Y., Kimura S., Davydov I. V., Kwon Y. T., RGS4 and RGS5 are in vivo substrates of the N-end rule pathway, 10.1073/pnas.0507533102
  69. Halaris Angelos, Plietz John, Agmatine : Metabolic Pathway and Spectrum of Activity in Brain, 10.2165/00023210-200721110-00002
  70. Dev. Biol., 112, 141 (2003)
  71. Maeda Hideki, Sahara Hiroeki, Mori Yoko, Torigo Toshihiko, Kamiguchi Kenjiro, Tamura Yutaka, Tamura Yasuaki, Hirata Kouichi, Sato Noriyuki, Biological Heterogeneity of the Peptide-binding Motif of the 70-kDa Heat Shock Protein by Surface Plasmon Resonance Analysis, 10.1074/jbc.m703436200
  72. J. Biol. Chem., 265, 15160 (1990)
  73. Ellis R. John, Minton Allen P., Cell biology: Join the crowd, 10.1038/425027a
  74. Minton Allen P., The Influence of Macromolecular Crowding and Macromolecular Confinement on Biochemical Reactions in Physiological Media, 10.1074/jbc.r100005200
  75. Chuang S.-M., Chen L., Lambertson D., Anand M., Kinzy T. G., Madura K., Proteasome-Mediated Degradation of Cotranslationally Damaged Proteins Involves Translation Elongation Factor 1A, 10.1128/mcb.25.1.403-413.2005
  76. Redd Michael J., Arnaud Martha B., Johnson Alexander D., A Complex Composed of Tup1 and Ssn6 Represses Transcriptionin Vitro, 10.1074/jbc.272.17.11193
  77. Malavé Tania M., Dent Sharon Y.R., Transcriptional repression by Tup1–Ssn6This paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process., 10.1139/o06-073
  78. Smith Rebecca L, Johnson Alexander D, Turning genes off by Ssn6–Tup1: a conserved system of transcriptional repression in eukaryotes, 10.1016/s0968-0004(00)01592-9
  79. Du F., Navarro-Garcia F., Xia Z., Tasaki T., Varshavsky A., Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain, 10.1073/pnas.172527399
  80. Xie Y., Varshavsky A., Physical association of ubiquitin ligases and the 26S proteasome, 10.1073/pnas.060025497
  81. Xie Y., Varshavsky A., RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit, 10.1073/pnas.071022298
  82. Ju Donghong, Xu Haiming, Wang Xiaogang, Xie Youming, Ubiquitin-mediated degradation of Rpn4 is controlled by a phosphorylation-dependent ubiquitylation signal, 10.1016/j.bbamcr.2007.04.012
  83. Catic André, Sun Zhen-Yu J, Ratner Daniel M, Misaghi Shahram, Spooner Eric, Samuelson John, Wagner Gerhard, Ploegh Hidde L, Sequence and structure evolved separately in a ribosomal ubiquitin variant, 10.1038/sj.emboj.7601772
  84. Lynch, M. (2007) The Origins of Genome Architecture, Sinauer Associates, Inc., Sunderland, MA
  85. Nasmyth Kim, Uhlmann Frank, Lottspeich Friedrich, 10.1038/21831
  86. Xie Y., The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain, 10.1093/emboj/18.23.6832
Bibliographic reference Xia, Zanxian ; Webster, Ailsa ; Du, Fangyong ; Piatkov, Konstantin ; Ghislain, Michel ; et. al. Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway.. In: The Journal of biological chemistry, Vol. 283, no. 35, p. 24011-28 (2008)
Permanent URL http://hdl.handle.net/2078.1/28222