The Substrate Recognition Domains of the N-end Rule Pathway
2008; Elsevier BV; Volume: 284; Issue: 3 Linguagem: Inglês
10.1074/jbc.m803641200
ISSN1083-351X
AutoresTakafumi Tasaki, Adriana Zakrzewska, Drew D. Dudgeon, Yonghua Jiang, John S. Lazo, Yong Tae Kwon,
Tópico(s)Protein Degradation and Inhibitors
ResumoThe N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway. The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway. The N-end rule pathway is a ubiquitin (Ub) 2The abbreviations used are: Ub, ubiquitin; degron, degradation signal; N-degron, N-terminal degron; MEF, mouse embryonic fibroblast; CECF, continuous-exchange cell-free; MBP, maltose-binding protein; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid.-dependent proteolytic system in which N-terminal residues of short-lived proteins function as an essential component of degradation signals (degrons) called N-degrons (Fig. 1A) (1Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 2Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (724) Google Scholar, 3Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (153) Google Scholar, 4Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (114) Google Scholar, 5Kwon Y.T. Lévy F. Varshavsky A. J. Biol. Chem. 1999; 274: 18135-18139Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Kwon 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. Mol. Cell. Biol. 2000; 20: 4135-4148Crossref PubMed Scopus (89) Google Scholar, 7Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (117) Google Scholar, 8Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (269) Google Scholar, 9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar, 10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar, 11Tasaki T. Sohr R. Xia Z. Hellweg R. Hortnagl H. Varshavsky A. Kwon Y.T. J. Biol. Chem. 2007; 282: 18510-18520Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 12Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (197) Google Scholar, 14Lee M.J. Pal K. Tasaki T. Roy S. Jiang Y. An J.Y. Banerjee R. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 100-105Crossref PubMed Scopus (43) Google Scholar, 15An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (73) Google Scholar). An N-degron can be created from a pre-N-degron through specific N-terminal modifications (12Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing residues that function through their deamidation by N-terminal amidohydrolases into the secondary destabilizing N-terminal residues Asp and Glu, respectively (6Kwon 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. Mol. Cell. Biol. 2000; 20: 4135-4148Crossref PubMed Scopus (89) Google Scholar, 16Grigoryev S. Stewart A.E. Kwon Y.T. Arfin S.M. Bradshaw R.A. Jenkins N.A. Copeland N.G. Varshavsky A. J. Biol. Chem. 1996; 271: 28521-28532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) (Fig. 1A). N-terminal Asp and Glu are secondary destabilizing residues that function through their arginylation by ATE1 R-transferase, which creates the primary destabilizing residue Arg at the N terminus (4Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (114) Google Scholar, 8Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (269) Google Scholar) (Fig. 1A). N-terminal Cys can also function as a tertiary destabilizing residue through its oxidation in a manner depending on nitric oxide and oxygen (O2); the oxidized Cys residue is subsequently arginylated by ATE1 (8Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (269) Google Scholar, 13Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (197) Google Scholar, 17Hu R.G. Sheng J. Qi X. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (243) Google Scholar). N-terminal Arg together with other primary destabilizing N-terminal residues are directly bound by specific E3 Ub ligases called N-recognins (3Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (153) Google Scholar, 7Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (117) Google Scholar, 9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar). Destabilizing N-terminal residues can be created through the removal of N-terminal Met or the endoproteolytic cleavage of a protein, which exposes a new amino acid at the N terminus (12Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (197) Google Scholar). N-terminal degradation signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2 (bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues (2Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (724) Google Scholar, 12Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In addition to a destabilizing N-terminal residue, a functional N-degron requires at least one internal Lys residue (the site of a poly-Ub chain formation) and a conformational feature required for optimal ubiquitylation (1Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 2Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (724) Google Scholar, 18Prakash S. Tian L. Ratliff K.S. Lehotzky R.E. Matouschek A. Nat. Struct. Mol. Biol. 2004; 11: 830-837Crossref PubMed Scopus (362) Google Scholar). UBR1 and UBR2 are functionally overlapping N-recognins (3Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (153) Google Scholar, 7Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (117) Google Scholar, 9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar). Our proteomic approach using synthetic peptides bearing destabilizing N-terminal residues captured a set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR box (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar, 11Tasaki T. Sohr R. Xia Z. Hellweg R. Hortnagl H. Varshavsky A. Kwon Y.T. J. Biol. Chem. 2007; 282: 18510-18520Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 12Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). UBR5 is a HECT E3 ligase known as EDD (E3 identified by differential display) (19Callaghan M.J. Russell A.J. Woollatt E. Sutherland G.R. Sutherland R.L. Watts C.K. Oncogene. 1998; 17: 3479-3491Crossref PubMed Scopus (95) Google Scholar) and a homolog of Drosophila hyperplastic discs (20Mansfield E. Hersperger E. Biggs J. Shearn A. Dev. Biol. 1994; 165: 507-526Crossref PubMed Scopus (77) Google Scholar). The mammalian genome encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7 (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar). UBR box proteins are generally heterogeneous in size and sequence but contain, with the exception of UBR4, specific signatures unique to E3s or a substrate recognition subunit of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7 (Fig. 2B). The biochemical properties of more recently identified UBR box proteins, such as UBR3 through UBR7, are largely unknown. Studies using knock-out mice implicated the N-end rule pathway in cardiac development and signaling, angiogenesis (8Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (269) Google Scholar, 15An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (73) Google Scholar), meiosis (9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar), DNA repair (21Ouyang Y. Kwon Y.T. An J.Y. Eller D. Tsai S.C. Diaz-Perez S. Troke J.J. Teitell M.A. Marahrens Y. Mut. Res. 2006; 596: 64-75Crossref PubMed Scopus (20) Google Scholar), neurogenesis (15An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (73) Google Scholar), pancreatic functions (22Zenker M. Mayerle J. Lerch M.M. Tagariello A. Zerres K. Durie P.R. Beier M. Hülskamp G. Guzman C. Rehder H. Beemer F.A. Hamel B. Vanlieferinghen P. Gershoni-Baruch R. Vieira M.W. Dumic M. Auslender R. Gil-da-Silva-Lopes V.L. Steinlicht S. Rauh M. Shalev S.A. Thiel C. Ekici A.B. Winterpacht A. Kwon Y.T. Varshavsky A. Reis A. Nat. Genet. 2005; 37: 1345-1350Crossref PubMed Scopus (195) Google Scholar), learning and memory (23Balogh S.A. Kwon Y.T. Denenberg V.H. Learn. Mem. 2000; 7: 279-286Crossref PubMed Scopus (26) Google Scholar, 24Balogh S.A. McDowell C.S. Kwon Y.T. Denenberg V.H. Brain Res. 2001; 892: 336-343Crossref PubMed Scopus (16) Google Scholar), female development (9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar), muscle atrophy (25Lecker S.H. Solomon V. Price S.R. Kwon Y.T. Mitch W.E. Goldberg A.L. J. Clin. Investig. 1999; 104: 1411-1420Crossref PubMed Scopus (152) Google Scholar), and olfaction (11Tasaki T. Sohr R. Xia Z. Hellweg R. Hortnagl H. Varshavsky A. Kwon Y.T. J. Biol. Chem. 2007; 282: 18510-18520Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Mutations in human UBR1 is a cause of Johanson-Blizzard syndrome (22Zenker M. Mayerle J. Lerch M.M. Tagariello A. Zerres K. Durie P.R. Beier M. Hülskamp G. Guzman C. Rehder H. Beemer F.A. Hamel B. Vanlieferinghen P. Gershoni-Baruch R. Vieira M.W. Dumic M. Auslender R. Gil-da-Silva-Lopes V.L. Steinlicht S. Rauh M. Shalev S.A. Thiel C. Ekici A.B. Winterpacht A. Kwon Y.T. Varshavsky A. Reis A. Nat. Genet. 2005; 37: 1345-1350Crossref PubMed Scopus (195) Google Scholar), an autosomal recessive disorder with multiple developmental abnormalities (26Johanson A. Blizzard R. J. Pediatr. 1971; 79: 982-987Abstract Full Text PDF PubMed Scopus (130) Google Scholar). Other functions of the pathway include: (i) a nitric oxide and oxygen (O2) sensor controlling the proteolysis of RGS4, RGS5, and RGS16 (8Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (269) Google Scholar, 13Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (197) Google Scholar, 17Hu R.G. Sheng J. Qi X. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (243) Google Scholar), (ii) a heme sensor through hemin-dependent inhibition of ATE1 function (27Hu R.G. Wang H. Xia Z. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 76-81Crossref PubMed Scopus (93) Google Scholar), (iii) the regulation of short peptide import through the peptide-modulated degradation of the repressor of the import (28Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (85) Google Scholar, 29Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (168) Google Scholar), (iv) the control of chromosome segregation through the degradation of a separate produced cohesin fragment (30Rao H. Uhlmann F. Nasmyth K. Varshavsky A. Nature. 2001; 410: 955-959Crossref PubMed Scopus (231) Google Scholar), (v) the regulation of apoptosis through the degradation of a caspase-processed inhibitor of apoptosis (31Ditzel M. Wilson R. Tenev T. Zachariou A. Paul A. Deas E. Meier P. Nat. Cell Biol. 2003; 5: 467-473Crossref PubMed Scopus (206) Google Scholar, 32Varshavsky A. Nat. Cell Biol. 2003; 5: 373-376Crossref PubMed Scopus (93) Google Scholar), (vi) the control of the human immunodeficiency virus replication cycle through the degradation of human immunodeficiency virus integrase (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar, 33Lloyd A.G. Ng Y.S. Muesing M.A. Simon V. Mulder L.C. Virology. 2007; 360: 129-135Crossref PubMed Scopus (20) Google Scholar), and (vii) the regulation of leaf senescence in plants (34Yoshida S. Ito M. Gallis J. Nishida I. Watanabe A. Plant J. 2002; 32: 129-137Crossref PubMed Scopus (121) Google Scholar). In the present study we characterized substrate binding specificities and recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4, and UBR5 were captured by N-terminal degradation determinants, whereas UBR3, UBR6, and UBR7 were not. We also report that in contrast to other E3 systems that usually recognize substrates through protein-protein interface, UBR1 and UBR2 have a general substrate recognition domain termed the UBR box. Remarkably, a 72-residue UBR box-only fragment fully retains its structural integrity and thereby the ability to recognize type-1 N-end rule substrates. We also report that the N-domain, structurally and functionally related with bacterial N-recognins, is required for recognizing type-2 N-end rule substrates. We discuss the evolutionary relationship between eukaryotic and prokaryotic N-recognins. Overexpression of UBR Proteins in Mammalian Cells—The plasmid pcDNA3flagUBR2 (9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar) was used to express N-terminal FLAG-tagged mouse UBR2 in COS7 cells from the PCMV promoter. The human UBR4 cDNA was excised from plasmid 7124A (a gift from Dr. Scott Vande Pol, University of Virginia) using SalI and NotI, and was subcloned into the plasmid pENTR3C (Invitrogen) to yield the entry vector pENTR3ChUBR4. By using the Gateway system (Invitrogen), the 15.9-kb UBR4 open reading frame was transferred from the pENTR3ChUBR4 to the destination vector pcDNA6.2/clumio-DEST (Invitrogen), yielding plasmid pcDNA6.2/cluvhUBR4 that expresses C-terminal lumio-V5-tagged UBR4 with a size of 570 kDa in COS7 cells from the PCMV promoter. Plasmids S503 and S3 (gifts from Drs. Michelle Henderson and CKW Watts, Garvan Institute of Medical Research) were used to express N-terminal FLAG-tagged full-length human UBR5 and its truncated derivative UBR5-(889-1877) in mammalian cells from PSV40 promoter. Cells were harvested 48 h after transfection, and cytosolic extracts were prepared as described (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar). Ubr1-/-, Ubr2-/-, Ubr1-/-Ubr2-/-, and Ubr1-/-Ubr2-/-Ubr4RNAi mouse embryonic fibroblasts (MEFs) have been previously established (7Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (117) Google Scholar, 9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar, 10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar). Protein Expression Using a Continuous-exchange Cell-free (CECF) System—Full-length UBR proteins or their fragments were expressed in vitro and labeled with [35S]methionine using the RTS 100 Wheat Germ CECF system (Roche, Germany) according to the manufacturer's protocol. Briefly, the PT7 promoter-based linear DNA templates were generated by using two-step PCR. DNA templates for the first PCR were m-Ubr1 (7Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (117) Google Scholar), m-Ubr2 (9Kwon Y.T. Xia Z. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Sheng J. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (124) Google Scholar), m-Ubr3 (11Tasaki T. Sohr R. Xia Z. Hellweg R. Hortnagl H. Varshavsky A. Kwon Y.T. J. Biol. Chem. 2007; 282: 18510-18520Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), h-UBR4 (7124A), h-UBR5 (S503), m-Ubr6 (cDNA clone IMAGE 4237432), and m-Ubr7 (cDNA clone IMAGE 6812389). Escherichia coli clps gene was amplified using genomic DNA from the strain DH5α cells (Invitrogen). The wheat germ lysate containing amino acids, RNA polymerases, DNA templates, and [35S]methionine (Amersham Bioscience) was incubated for 24 h at 25 °C in the CECF unit. Glycerol (50 μl) was added to the reaction mixture (50 μl) to stabilize expressed proteins. To evaluate the level of synthesized proteins, the incorporated 35S was counted using trichloroacetic acid precipitation. Synthesized proteins were stored at -20 °C and used for assays within 3 days. Proteins were also expressed using the transcription-translation-coupled TnT system (Promega, Madison, WI) as described (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar). The X-peptide Pull-down Assay—For the X-peptide pull-down assay, a set of 12-mer peptides (X-peptides) bearing N-terminal Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residues were cross-linked through the C-terminal Cys residue to Ultralink Iodoacetyl beads (Pierce) as described previously (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar) (Fig. 1B, left). The ratio of peptides to beads was ∼1 μmol of peptides per 1-ml beads. Alternatively, the otherwise identical 12-mer peptides, bearing C-terminal biotinylated Lys instead of Cys, were conjugated, via biotin, to streptavidin-Sepharose beads (Amersham Bioscience) to a ratio of 1-1.5 μmol of peptides per 1 ml of beads (Fig. 1B, right). RTS wheat germ lysates (50 μl) expressing 35S-labeled proteins were diluted 2-fold and centrifuged to remove precipitates. An aliquot (10-20 μl) of soluble extract, containing 50-100 μg of total protein, was diluted in 250 μl of binding buffer A (0.1% Nonidet P-40, 10% glycerol, 0.15 m KCl, and 20 mm HEPES, pH 7.9) and mixed with X-peptide beads (7.5-μl packed volume for cross-linked peptide beads or 10-μl packed volume for biotinylated peptide beads). The mixtures were incubated at 4 °C for 4 h with a gentle rotation in the presence or absence of dipeptides. The beads were pelleted by centrifugation at 2,400 × g for 30 s, washed three times with 0.25 ml of binding buffer A, resuspended in 15 μl of SDS-PAGE sample buffer, and heated at 55 °C for 30 min. To analyze the binding property of endogenous UBR proteins, extracts from mouse testes were prepared and subjected to the X-peptide pull-down assay essentially as described (10Tasaki T. Mulder L.C. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (247) Google Scholar). COS7 cell extracts expressing UBR proteins were diluted to ∼1.0 mg/ml in binding buffer A for pull-down assays. Denatured proteins in SDS-PAGE sample buffer were separated in 10% BisTris acrylamide gel with MES buffer, followed by fixing with solution A (50% methanol and 10% acetic acid in water) for 20 min and subsequently with solution B (25% methanol and 10% acetic acid in water) for 10 min. The 35S-labeled proteins were detected using autoradiography or, for quantitation, using a PhosphorImager (Bio-Rad). Site-directed Mutagenesis—Site-directed mutagenesis was employed to express 31 mutant 50-kDa UBR1-(1-453) proteins, each of which contained a mutation to alanine (Ala), by using overlap extension PCR (35Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). Two first round PCRs, with primers containing mismatch nucleotides, generated two DNA fragments with overlap extension of 15-24 bp. PCR primer sequences are available upon request. The second round PCR, using two DNA fragments from the first round PCRs as templates, yielded a single chimeric DNA fragment containing a mutation to Ala. The resulting PCR fragments were used as templates in the third PCR to generate the PT7 promoter-based linear DNA templates for the RTS protein expression system. The final mutant DNA fragments were evaluated by sequencing. Bacterial Expression and Purification of MBP-UBR1-(91-191)-His6 and MBP-His6—A cDNA fragment encoding C-terminal His6-tagged UBR1-(91-191) was subcloned into a bacterial expression vector pMAL-c4X (New England Biolabs, Boston, MA) that expresses maltose-binding protein (MBP). The resulting plasmid pMalUBRbox expresses MBP-UBR1-(91-191)-His6. MBP-UBR1-(91-191)-His6 and its control, MBP-His6, were overexpressed in E. coli BL21(DE3) cells and subjected to two-step purification using amylose-resin column chromatography (New England Biolabs) and cobalt-Sepharose column chromatography (Pierce). Purified proteins (5 μg) were subjected to the X-peptide pull-down assay for 1 h in the binding buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 0.05% Tween 20). Surface Plasmon Resonance (Biacore) Assay—The direct surface plasmon resonance was measured using a Biacore T100 biosensor (GE Healthcare). Prior to peptide loading, the surface of the SA sensor chip was conditioned by 4-5 washes of 1 m NaCl and 50 mm NaOH at a flow rate of 30 μl/min for 30 s to wash off nonspecifically or poorly bound streptavidin. Biotinylated Arg-peptide, Phe-peptide, and Gly-peptide, adjusted to 20 nm in the binding buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, and 0.05% P20 surfactant), were immobilized on the surface of the SA sensor chip at a flow rate of 20 μl/min to reach ∼200 response units. Purified MBP-UBR1-(91-191)-His6 and MBP-His6, adjusted to 5 μm in the binding buffer, were injected at a flow rate of 40 μl/min. After dissociation for 60 s, the surface was regenerated back to bound peptides by a 30-s injection of 50 mm EDTA at a flow rate of 10 μl/min. Kinetics experiments were performed using MBP-UBR1-(91-191)-His6 at varying concentrations (0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25, 2.5, and 3.13 μm). A duplicate concentration at 3.13 μm was used to determine cycle to cycle variability, and all data were double-referenced to a buffer-only control and a reference flow cell without biotinylated peptides. The data were fit using a 1:1 interaction model where A + B = AB, including terms for mass transport, using Biacore T100 Evaluation Software version 1.1.1. Binding Properties of UBR Proteins to Destabilizing N-terminal Residues—We employed X-peptide pull-down assays to characterize the binding specificities of UBR proteins for destabilizing N-terminal residues. X-peptide beads (X = Arg (type 1), Phe (type 2), or Gly (stabilizing control)) (Fig. 1B) were mixed with mouse testes extracts and precipitated by centrifugation, followed by immunoblotting to detect the presence of UBR proteins in precipitates. Arg-peptide captured endogenous UBR1, UBR2, UBR4, and UBR5 from testes extracts, whereas Phe-peptide brought down UBR1, UBR2, and UBR4, but not UBR5 (Fig. 1C, data not shown). None of these UBR proteins were detected in precipitates prepared by Gly-peptide. Thus, we confirmed that endogenous UBR1, UBR2, UBR4, and UBR5 can be captured by destabilizing N-terminal residues using a different experimental setting. (Because either method for peptide-bead conjugation, through C-terminal Cys or biotin, gave essentially the same results, the conjugation method is not specified in subsequent experiments.) The binding of UBR1 (Fig. 1D), UBR2, and UBR4 (data not shown) to the type-2 substrate Phe was not inhibited by increasing salt (NaCl) concentrations up to 1.0 m. This result indicates that type-2 substrate recognition involves hydrophobic interaction, consistent with type-2 N termini (Phe, Leu, Trp, Tyr, and Ile) being bulky “hydrophobic.” In contrast, the binding of UBR1 and UBR5 to the type-1 substrate Arg was significantly affected by salt concentrations at a range of 0.15-0.5 m (Fig. 1D), in agreement with type-1 substrates (Arg, Lys, and His) being “basic.
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