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• W2100524871 abstract "Arf GTPases control vesicle formation from different intracellular membranes and are regulated by Arf guanine nucleotide exchange factors (GEFs). Outside of their conserved catalytic domains, known as Sec7 domains, little is known about Arf GEFs. Rsp5 is a yeast ubiquitin ligase that regulates numerous membrane trafficking events and carries a C2 domain that is specifically required for trans-Golgi network to vacuole transport. In a screen for proteins that interact with the Rsp5 C2 domain we identified Sec7, the GEF that acts on Golgi-associated Arfs. The Rsp5-Sec7 interaction is direct, occurs in vivo, and is conserved among mammalian Rsp5 and Sec7 homologues. A 50-amino acid region near the Sec7 C terminus is required for Rsp5 binding and for normal Sec7 localization. Binding of Sec7 to Rsp5 is dependent on the presence of the phosphoinositide 3-kinase Vps34, suggesting that phosphatidylinositol 3-phosphate (PI(3)P) plays a role in regulating this interaction. Overexpression of Sec7 significantly suppresses the growth and sorting defects of an rsp5 C2 domain point mutant. These observations identify a new functional region within the Sec7/BIG family of Arf GEFs that is required for trans-Golgi network localization. Arf GTPases control vesicle formation from different intracellular membranes and are regulated by Arf guanine nucleotide exchange factors (GEFs). Outside of their conserved catalytic domains, known as Sec7 domains, little is known about Arf GEFs. Rsp5 is a yeast ubiquitin ligase that regulates numerous membrane trafficking events and carries a C2 domain that is specifically required for trans-Golgi network to vacuole transport. In a screen for proteins that interact with the Rsp5 C2 domain we identified Sec7, the GEF that acts on Golgi-associated Arfs. The Rsp5-Sec7 interaction is direct, occurs in vivo, and is conserved among mammalian Rsp5 and Sec7 homologues. A 50-amino acid region near the Sec7 C terminus is required for Rsp5 binding and for normal Sec7 localization. Binding of Sec7 to Rsp5 is dependent on the presence of the phosphoinositide 3-kinase Vps34, suggesting that phosphatidylinositol 3-phosphate (PI(3)P) plays a role in regulating this interaction. Overexpression of Sec7 significantly suppresses the growth and sorting defects of an rsp5 C2 domain point mutant. These observations identify a new functional region within the Sec7/BIG family of Arf GEFs that is required for trans-Golgi network localization. Newly synthesized proteins are transported from the Golgi to either the plasma membrane or the lysosome. This sorting decision is made at the trans-Golgi network (TGN) 5The abbreviations used are: TGN, trans-Golgi network; GFP, green fluorescent protein; CIR, C2 domain interacting region; GEF, guanine nucleotide exchange factor; PI(3)P, phosphatidylinositol 3-phosphate; GST, glutathione S-transferase; CPS, carboxypeptidase S; MVE, multivesicular endosome; PI 3-kinase, phosphoinositide 3-kinase; aa, amino acid; IP, immunoprecipitation; MES, 4-morpholineethanesulfonic acid. 5The abbreviations used are: TGN, trans-Golgi network; GFP, green fluorescent protein; CIR, C2 domain interacting region; GEF, guanine nucleotide exchange factor; PI(3)P, phosphatidylinositol 3-phosphate; GST, glutathione S-transferase; CPS, carboxypeptidase S; MVE, multivesicular endosome; PI 3-kinase, phosphoinositide 3-kinase; aa, amino acid; IP, immunoprecipitation; MES, 4-morpholineethanesulfonic acid. where separation of secreted proteins from lysosomal proteins occurs. In mammalian cells sorting at the TGN targets proteins for secretion, to apical or basolateral membranes, or to the lysosome. In yeast, proteins follow similar pathways, although the distinction between plasma membrane compartments does not exist and the intracellular destination is the lysosome-like vacuole. Many proteins involved in these trafficking pathways have been identified and characterized in yeast and are conserved in mammalian cells (reviewed in Refs. 1Gu F. Crump C.M. Thomas G. Cell Mol. Life Sci. 2001; 58: 1067-1084Crossref PubMed Scopus (140) Google Scholar, 2Rodriguez-Boulan E. Musch A. Biochim. Biophys. Acta. 2005; 1744: 455-464Crossref PubMed Scopus (103) Google Scholar, 3Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (135) Google Scholar). Some integral membrane cargos destined for the lysosome are modified with a monoubiquitin signal at the TGN by members of the Nedd4/Rsp5 family of ubiquitin ligases (reviewed in Refs. 4Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (960) Google Scholar and 5Shearwin-Whyatt L. Dalton H.E. Foot N. Kumar S. Bioessays. 2006; 28: 617-628Crossref PubMed Scopus (132) Google Scholar). Other integral membrane and luminal proteins do not require modification with ubiquitin for efficient trafficking from the TGN to the endosomal sorting pathway. However, the role of ubiquitin in sorting of these cargos within the endosomal pathway remains unclear (6Pak Y. Glowacka W.K. Bruce M.C. Pham N. Rotin D. J. Cell Biol. 2006; 175: 631-645Crossref PubMed Scopus (78) Google Scholar, 7McNatt M.W. McKittrick I. West M. Odorizzi G. Mol. Biol. Cell. 2007; 18: 697-706Crossref PubMed Scopus (65) Google Scholar, 8Oestreich A.J. Aboian M. Lee J. Azmi I. Payne J. Issaka R. Davies B.A. Katzmann D.J. Mol. Biol. Cell. 2007; 18: 707-720Crossref PubMed Scopus (56) Google Scholar, 9Stawiecka-Mirota M. Pokrzywa W. Morvan J. Zoladek T. Haguenauer-Tsapis R. Urban-Grimal D. Morsomme P. Traffic. 2007; 8: 1280-1296Crossref PubMed Scopus (61) Google Scholar, 10Watson H. Bonifacino J.S. Mol. Biol. Cell. 2007; 18: 1781-1789Crossref PubMed Scopus (27) Google Scholar). Ubiquitinated cargo is recognized at the Golgi by the ubiquitin-binding domains in GGA (Golgi localized, ;-ear containing, Arf-binding) proteins that sort TGN cargo into vesicles (reviewed in Ref. 3Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (135) Google Scholar). After leaving the TGN, cargo travels to the lysosome through a late endosomal compartment, known as the multivesicular endosome (MVE), where monoubiquitinated cargo is sorted into vesicles that bud into the MVE lumen. The MVE vesicles and their contents are then delivered in their entirety into the lumen of the lysosome (reviewed in Refs. 11Piper R.C. Katzmann D.J. Annu. Rev. Cell Dev. Biol. 2007; 23: 519-547Crossref PubMed Scopus (523) Google Scholar and 12Hurley J.H. Curr. Opin. Cell Biol. 2008; 20: 4-11Crossref PubMed Scopus (342) Google Scholar). Arf proteins are the small GTPases that regulate vesicle formation at the TGN (13Moss J. Vaughan M. J. Biol. Chem. 1998; 273: 21431-21434Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 14Donaldson J.G. Biochem. Soc. Trans. 2005; 33: 1276-1278Crossref PubMed Scopus (36) Google Scholar). Like other GTPases, Arf proteins are activated upon exchange of bound GDP for GTP in a reaction catalyzed by guanine nucleotide exchange factors (GEFs). Arf activation by GEFs is a crucial regulatory step, because Arf must be in the GTP-bound state to recruit vesicle coat proteins to initiate vesicle budding (15Zhu Y. Traub L.M. Kornfeld S. Mol. Biol. Cell. 1998; 9: 1323-1337Crossref PubMed Scopus (88) Google Scholar, 16Takatsu H. Yoshino K. Toda K. Nakayama K. Biochem. J. 2002; 365: 369-378Crossref PubMed Scopus (95) Google Scholar). Arf GEFs are characterized by having a catalytic Sec7 domain, first identified in the Saccharomyces cerevisiae Sec7 protein (reviewed in Refs. 17Jackson C.L. Casanova J.E. Trends Cell Biol. 2000; 10: 60-67Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar and 18Casanova J.E. Traffic. 2007; 8: 1476-1485Crossref PubMed Scopus (272) Google Scholar). Sec7 is a large, essential, Golgi-associated Arf GEF required for the formation of transport vesicles from the TGN in yeast (19Achstetter T. Franzusoff A. Field C. Schekman R. J. Biol. Chem. 1988; 263: 11711-11717Abstract Full Text PDF PubMed Google Scholar). It is localized primarily to TGN membranes (20Franzusoff A. Lauze E. Howell K.E. Nature. 1992; 355: 173-175Crossref PubMed Scopus (48) Google Scholar). Mutations in Sec7 cause defects in the transport of a variety of cargo proteins from the Golgi to the vacuole, including proteins that travel by ubiquitin-independent and -dependent pathways (21Deitz S.B. Rambourg A. Kepes F. Franzusoff A. Traffic. 2000; 1: 172-183Crossref PubMed Scopus (33) Google Scholar, 22Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar). Sec7 has two mammalian homologues, BIG1 and BIG2, that primarily localize to the TGN to function in the release of vesicles from this organelle (reviewed in Ref. 23Shin H.W. Nakayama K. J. Biochem. (Tokyo). 2004; 136: 761-767Crossref PubMed Scopus (59) Google Scholar). BIG2 also localizes to the recycling endosome and is involved in organelle integrity (24Shin H.W. Morinaga N. Noda M. Nakayama K. Mol. Biol. Cell. 2004; 15: 5283-5294Crossref PubMed Scopus (106) Google Scholar). Beyond the central catalytic domain, there are no characterized domains within Sec7, and little is known about the interactions and functions of the N- and C-terminal regions of the protein (21Deitz S.B. Rambourg A. Kepes F. Franzusoff A. Traffic. 2000; 1: 172-183Crossref PubMed Scopus (33) Google Scholar). Recently, regions of homology have been identified within BIG1 and BIG2 (reviewed in Ref. 18Casanova J.E. Traffic. 2007; 8: 1476-1485Crossref PubMed Scopus (272) Google Scholar). These regions are sites of dimerization between BIG proteins and also interact with other binding partners (25Li H. Adamik R. Pacheco-Rodriguez G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1627-1632Crossref PubMed Scopus (70) Google Scholar, 26Xu K.F. Shen X. Li H. Pacheco-Rodriguez G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2784-2789Crossref PubMed Scopus (44) Google Scholar, 27Ishizaki R. Shin H.W. Iguchi-Ariga S.M. Ariga H. Nakayama K. Genes Cells. 2006; 11: 949-959Crossref PubMed Scopus (22) Google Scholar, 28Ramaen O. Joubert A. Simister P. Belgareh-Touze N. Olivares-Sanchez M.C. Zeeh J.C. Chantalat S. Golinelli-Cohen M.P. Jackson C.L. Biou V. Cherfils J. J. Biol. Chem. 2007; 282: 28834-28842Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 29Ishizaki R. Shin H.W. Mitsuhashi H. Nakayama K. Mol. Biol. Cell. 2008; 19: 2650-2660Crossref PubMed Scopus (70) Google Scholar). However, the function of these interactions is still unclear. Ubiquitin signals are attached to biosynthetic cargo traveling from the TGN to the lysosome by ubiquitin ligases, regulatory proteins that catalyze the transfer of ubiquitin to substrates (reviewed in Refs. 30Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar and 31d'Azzo A. Bongiovanni A. Nastasi T. Traffic. 2005; 6: 429-441Crossref PubMed Scopus (202) Google Scholar). One family of ubiquitin ligases that plays important roles in a variety of membrane trafficking processes is named for the mammalian Nedd4 and yeast Rsp5 proteins. Rsp5 is the sole S. cerevisiae member of the Nedd4/Rsp5 family; mammalian members include Nedd4–1, Nedd4–2, WWP1/AIP5, WWP2/AIP2, AIP4/Itch, Smurf1, and Smurf2. These ligases have a conserved domain structure, an N-terminal C2 domain, two to four central WW domains, and a C-terminal HECT (homologous to E6AP C terminus) catalytic domain (5Shearwin-Whyatt L. Dalton H.E. Foot N. Kumar S. Bioessays. 2006; 28: 617-628Crossref PubMed Scopus (132) Google Scholar, 30Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar, 32Ingham R.J. Gish G. Pawson T. Oncogene. 2004; 23: 1972-1984Crossref PubMed Scopus (392) Google Scholar). The function of the C2 domains in these ligases is ambiguous. C2 domains are found in proteins involved in vesicle trafficking, lipid modification, GTPase regulation, and protein phosphorylation (33Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (688) Google Scholar, 34Rizo J. Súdhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 35Cho W. Stahelin R.V. Biochim. Biophys. Acta. 2006; 1761: 838-849Crossref PubMed Scopus (213) Google Scholar). They bind to both phospholipids and proteins, and binding of Ca2+ to C2 domains can regulate these interactions (34Rizo J. Súdhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 36Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The C2 domains of Nedd4 and Smurf2 are important for interaction of these proteins with the plasma membrane (37Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 38Plant P.J. Lafont F. Lecat S. Verkade P. Simons K. Rotin D. J. Cell Biol. 2000; 149: 1473-1484Crossref PubMed Scopus (123) Google Scholar, 39Vecchione A. Marchese A. Henry P. Rotin D. Morrione A. Mol. Cell Biol. 2003; 23: 3363-3372Crossref PubMed Scopus (209) Google Scholar, 40Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar, 41Suzuki C. Murakami G. Fukuchi M. Shimanuki T. Shikauchi Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; 277: 39919-39925Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The Smurf2 C2 domain also plays an autoinhibitory function through binding to the HECT domain that inhibits autoubiquitination and substrate ubiquitination (42Wiesner S. Ogunjimi A.A. Wang H.R. Rotin D. Sicheri F. Wrana J.L. Forman-Kay J.D. Cell. 2007; 130: 651-662Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The Rsp5 C2 domain regulates trafficking in the late endocytic pathway at the MVE (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 44Katzmann D.J. Sarkar S. Chu T. Audhya A. Emr S.D. Mol. Biol. Cell. 2004; 15: 468-480Crossref PubMed Scopus (122) Google Scholar, 45Morvan J. Froissard M. Haguenauer-Tsapis R. Urban-Grimal D. Traffic. 2004; 5: 383-392Crossref PubMed Scopus (60) Google Scholar). Previously, we demonstrated that the Rsp5 C2 domain binds phosphoinositides (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar), particularly PI(3)P, which is enriched on endosomal membranes (reviewed in Ref. 46Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (240) Google Scholar). Specific amino acids within the Rsp5 C2 domain that are required for PI(3)P binding are also required for the ubiquitination of cargo traveling from the TGN to the MVE, and thus for the sorting of this cargo into MVE vesicles (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar). To further understand the function of the Rsp5 C2 domain in TGN to vacuole trafficking, we screened for proteins that bind to and function with the C2 domain. Here we identify the Arf GEF Sec7 as an Rsp5 C2 domain-binding protein in vitro and in vivo, and we investigate the physical and functional characteristics of this interaction. Strains, Media, and Reagents—Strains used in this study are listed in Table 1. Yeast strains were propagated in rich medium (2% Bacto-peptone, 1% yeast extract, 2% glucose supplemented with 20 mg/liter adenine, uracil, and tryptophan) or selective minimal medium (47Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). Caffeine-containing medium was prepared by adding caffeine to a final concentration of 6 mm before autoclaving.TABLE 1Yeast strainsStrainGenotypeaAll strains are MATa unless otherwise notedPJ69-4Atrp1–901 leu2–3,112 ura3–52 his3–200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZPJ69-4;MAT;, same as PJ69-4ALHY1107pNotI-RSP5[TRP1] rsp5Δ::HIS3 his3 trp1 lys2 ura3 leu2 bar1LHY1850ura3Δ0 leu2Δ0 his3Δ1 met15Δ0LHY3876prsp5K44,45,75,77,78Q[TRP1] pGFP-CPS rsp5Δ::HIS3 his3 leu2 ura3 trp1 bar1LHY3923prsp5K44,45,75,77,78Q[TRP1] rsp5Δ::HIS3 his3 leu2 ura3 trp1 bar1LHY4007prps5K75,77,78Q[TRP1] his3 trp1 lys2 ura3 leu2 bar1LHY4377pRSP5[TRP1] rsp5Δ::HIS3 leu2 ura3 trp1 bar1LHY4488pGFP-C2[TRP1] his3 trp1 lys2 ura3 leu2 bar1LHY5440SEC7-GFP::URA3 ura3Δ0 leu2Δ0 his3Δ1 met15Δ0LHY5466pNotI-RSP5[TRP1] rsp5Δ::HIS3 SEC7-GFP::URA3 his3 trp1 lys2 ura3 leu2 bar1LHY5467pNotI-rsp5ΔC2[TRP1] rsp5Δ::HIS3 SEC7-GFP::URA3 his3 trp1 lys2 ura3 leu2 bar1/bar1::HIS3LHY5474prsp5K44,45,75,77,78Q[TRP1] pSEC7[URA3] rsp5Δ::HIS3 his3 leu2 ura3 trp1 bar1LHY5476prsp5K44,45,75,77,78Q[TRP1] rsp5Δ::HIS3 his3 leu2 ura3 trp1 bar1LHY5508vps34Δ::kanMX4 SEC7-GFP::URA3 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0LHY5512prsp5K44,45,75,77,78Q[TRP1] pSEC7[LEU2] pGFP-CPS[URA3] rsp5Δ::HIS3 his3 leu2 ura3 trp1 bar1LHY5518pNotI-RSP5[TRP1] rsp5Δ::HIS3 sec7ΔCIR-GFP::URA3 his3 trp1 lys2 ura3 leu2 bar1LHY5519pNotI-rsp5ΔC2[TRP1] rsp5Δ::HIS3 sec7ΔCIR-GFP::URA3 his3 trp1 lys2 ura3 leu2 bar1/bar1::HIS3LHY5521pRSP5[TRP1] pGFP-CPS[URA3] rsp5Δ::HIS3 leu2 ura3 trp1 bar1LHY5604pNotI-RSP5[TRP1] rsp5Δ::HIS3 Sec7::URA3 SYS1-GFP::LEU2 his3 trp1 lys2 ura3 leu2 bar1LHY5605pNotI-RSP5[TRP1] rsp5Δ::HIS3 sec7ΔCIR::URA3 SYS1-GFP::LEU2 his3 trp1 lys2 ura3 leu2 bar1a All strains are MATa unless otherwise noted Open table in a new tab Anti-green fluorescent protein (GFP) antibodies were purchased from Roche Applied Sciences and anti-glutathione S-transferase (GST) antibodies were purchased from Amersham Biosciences. Anti-Rsp5 antiserum was previously described (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 48Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (119) Google Scholar). Plasmids—A multicopy SEC7 plasmid (YepTA65) (19Achstetter T. Franzusoff A. Field C. Schekman R. J. Biol. Chem. 1988; 263: 11711-11717Abstract Full Text PDF PubMed Google Scholar, 49Jones S. Jedd G. Kahn R.A. Franzusoff A. Bartolini F. Segev N. Genetics. 1999; 152: 1543-1556Crossref PubMed Google Scholar) was provided by N. Segev (University of Illinois, Chicago, IL). Plasmids encoding Sys1-GFP and Gap1-GFP were provided by B. Glick (University of Chicago, Chicago, IL) and R. Piper (University of Iowa, Iowa City, IA), respectively. URA3-marked or LEU2-marked plasmids encoding GFP-carboxypeptidase S (CPS) were provided by S. Emr (University of California, San Diego, CA) or generated in our laboratory. 6D. A. Klos Dehring, W. Lin, and L. Hicke, unpublished data. All site-directed mutagenesis was performed with a QuikChange® mutagenesis kit (Stratagene, La Jolla, CA). The yeast two-hybrid C2 domain bait vector (LHP2103) was constructed by PCR amplification of DNA encoding aa 1–142 of Rsp5, followed by insertion of the amplified fragment into the yeast two-hybrid GAL4-binding domain vector, pAS2-1 (Clontech, Palo Alto, CA). The 3K→Q (K75Q, K77Q, K78Q) mutation was introduced into the bait plasmid by site-directed mutagenesis (LHP2169). DNA encoding aa 1836–2009 of Sec7 was amplified from yeast genomic DNA and inserted into the pET-30 bacterial expression vector by ligation-independent cloning (Novagen, Madison, WI). Truncations and deletions of this fragment were introduced by site-directed mutagenesis. The same method was used to construct plasmids encoding the C termini of BIG1 (aa 1665–1849, LHP2396), BIG2 (aa 1567–1785, LHP2378), and Gea1 (aa 1352–1408, LHP2353). Bacterial expression plasmids encoding GST-C2, GST-Sla1350–420, and GST-Rvs167SH3 have been described (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 50Stamenova S.D. Dunn R. Adler A.S. Hicke L. J. Biol. Chem. 2004; 279: 16017-16025Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The 3K→Q mutation was generated in GST-C2 (LHP1665) by site-directed mutagenesis. DNA encoding the Itch C2 domain (aa 6–146, LHP2699) was amplified with terminal BglII sites and ligated into BamHI-digested pGEX-6P-2 (GE Healthcare). To construct an integrating plasmid for insertion of a C-terminal GFP tag into chromosomal SEC7, we amplified a fragment encoding Sec7 aa 1513–2009 from genomic DNA. This fragment was ligated into pUSE-URA3 (51Seron K. Tieaho V. Prescianotto-Baschong C. Aust T. Blondel M.-O. Guillaud P. Devilliers G. Rossanese O.W. Glick B.S. Riezman H. Keranen S. Haguenauer-Tsapis R. Mol. Biol. Cell. 1998; 9: 2837-2889Crossref Scopus (77) Google Scholar), a plasmid encoding aa 1815–2009 of Sec7 fused to a GFP, provided by B. Glick (University of Chicago, Chicago, IL). The endogenous SpeI site was removed from the resulting plasmid and another SpeI site was introduced with silent mutations at the codons for aa 1722 and 1723 (LHP2505). An integrating plasmid to construct a chromosomally encoded GFP-tagged Sec7 lacking the C2 domain-interacting region, GFP-Sec7ΔCIR, was similarly made with the additional removal of DNA encoding aa 1836–1883 (LHP2507). To provide wild-type and sec7 mutant strains for the localization of Sys1-GFP, we constructed untagged SEC7 and sec7ΔCIR integrants. DNA encoding the GFP tag from LHP2505 and LHP2507 was removed by digestion with BamHI and EagI. The ends of the remaining large fragments of the plasmids were converted to blunt ends with T4 DNA Polymerase (New England Biolabs, Beverly, MA) and the plasmids were ligated. A double stop codon was introduced after the codon for amino acid 2009 by site-directed mutagenesis resulting in plasmids encoding DNA to integrate untagged SEC7 (LHP2644) and untagged sec7ΔCIR (LHP2645). Plasmids were digested with SpeI and transformed into yeast. Replacement of the endogenous copy of SEC7 by homologous recombination was verified by PCR amplification of genomic DNA recovered from transformants. All mutations and plasmid sequences were verified by digestion and/or automated sequencing. Yeast Two-hybrid Screen—A plasmid encoding the binding domain of Gal4 fused to the Rsp5 C2 domain (LHP2103) was used to screen a yeast two-hybrid plasmid library (52James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). The yeast strain PJ69-4; was transformed with the yeast two-hybrid library and the strain PJ69-4A was transformed with LHP2103. The resulting transformants were mated and diploids were selected and transferred to minimal medium lacking leucine, tryptophan, and adenine to select colonies exhibiting a positive interaction. Plasmids from these colonies were recovered and co-transformed with the original bait plasmid (LHP2103) and a bait plasmid carrying the 3K→Q mutations (LHP2169). Plasmids from colonies that exhibited a positive interaction with the wild-type bait plasmid a second time were sequenced. Recombinant Protein Purification and Binding Experiments— Hexahistidine (His6)-tagged and GST-tagged recombinant proteins were expressed in BL21-Codon Plus Escherichia coli (Stratagene) propagated in Luria broth supplemented with 40 mg/ml kanamycin, or with 100 mg/ml ampicillin and 20 mg/ml chloramphenicol, for plasmid maintenance. Recombinant protein expression was induced at 18 or 24 °C with 1 mm isopropyl ;-d-thiogalactopyranoside (Sigma). Purification of His6-tagged proteins, purification of GST-tagged proteins, and binding experiments with these proteins were performed as previously described (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 50Stamenova S.D. Dunn R. Adler A.S. Hicke L. J. Biol. Chem. 2004; 279: 16017-16025Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 53Shih S.C. Katzmann K.J. Schnell J.D. Sutanto M. Emr S.C. Hicke L.H. Nat. Cell Biol. 2002; 4: 389-393Crossref PubMed Scopus (360) Google Scholar). Lysates and bound proteins were resolved by SDS-PAGE and analyzed by Coomassie staining or immunoblotting with anti-GST as previously described (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 48Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (119) Google Scholar). For binding experiments with GFP-tagged C2 domain (GFP-C2) in yeast lysates, cells were harvested at a density of 1–2 × 107 cells/ml. Lysates were prepared by mechanical agitation with glass beads in MES buffer (1% Triton X-100, 100 mm MES, pH 6.5, 0.5 mm MgCl2, 1 mm EGTA, 0.2 mm dithiothreitol) containing protease inhibitor mixture (0.2 ;g/ml chymostatin, 1 ;g/ml leupeptin, 2.5 ;g/ml antipain, 1 ;g/ml pepstatin, 1 mm phenylmethanesulfonyl fluoride). Lysates were incubated on ice for 1 h with 2 mg/ml ;-d-maltoside and 1% Triton X-100, cleared by centrifugation, and incubated with immobilized proteins as described for bacterial lysates. Lysates and bound proteins were analyzed by immunoblotting with anti-GFP as previously described (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 48Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (119) Google Scholar). Native Co-immunoprecipitation Experiments—Cells (1.5 × 109) were harvested at a density of 1–2 × 107 cells/ml. Lysates for immunoprecipitation were prepared by mechanical agitation with glass beads in GFP IP buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate) containing protease inhibitor mixture, 2 mg/ml ;-d-maltoside, and 1% Triton X-100. Lysates were incubated on ice for 1 h and cleared by centrifugation. Immunoprecipitations were performed overnight at 4 °C with GFP antibodies and Protein G-Sepharose (Amersham Biosciences). Precipitated proteins were washed with GFP IP buffer and GFP IP wash buffer (50 mm Tris-HCl, pH 7.5, 0.25 m NaCl, 0.1% Nonidet P-40, 0.05% deoxycholate), resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Sec7-GFP) or nitrocellulose membranes (Rsp5), and analyzed by immunoblotting as previously described using anti-Rsp5 or anti-GFP (43Dunn R. Klos D.A. Adler A.S. Hicke L. J. Cell Biol. 2004; 165: 135-144Crossref PubMed Scopus (121) Google Scholar, 48Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (119) Google Scholar). Fluorescence Microscopy—Cells were grown to a density of 1–2 × 107 cells/ml and harvested at 4 °C. Cells were embedded in 1.67% low melt agarose (American Biorganics Inc., Niagra Falls, NY) on a slide and analyzed by fluorescence microscopy (Leica DMIRE2 or Zeiss Axiovert 200M). For quantification of GFP-CPS sorting, cells with a detectable GFP signal were counted and binned into one of three groups based on the location of the GFP signal: group 1, vacuolar lumen (solid spot); group 2, limiting membrane of the vacuole (ring around the vacuole); or group 3, vacuolar lumen and limiting membrane (ring around the vacuole with diffuse internal fluorescence). For Sec7-GFP and Sys1-GFP images, cross-sections were taken along the z axis and projections were made using ImageJ version 1.34. Growth Suppression Analysis—Cells were transformed with an URA3-marked multicopy plasmid encoding SEC7 (YepTA65, Refs. 19Achstetter T. Franzusoff A. Field C. Schekman R. J. Biol. Chem. 1988; 263: 11711-11717Abstract Full Text PDF PubMed Google Scholar and 49Jones S. Jedd G. Kahn R.A. Franzusoff A. Bartolini F. Segev N. Genetics. 1999; 152: 1543-1556Crossref PubMed Google Scholar). To remove the plasmid, cells were grown on medium containing 5-fluoroorotic acid. For serial dilution growth assays, multiple transformants were grown overnight to stationary phase. Cells were serially diluted to 2 × 106, 2 × 105, 2 × 104, or 2 × 103 cells/ml and transferred in duplicate with an inoculating manifold to rich medium with or without 6 mm caffeine. Cells were grown at 37 °C. The Rsp5 C2 Domain Binds to the Sec7 C Terminus—C2 domains bind to both lipids and proteins (38Plant P.J. Lafont F. Lecat S. Verkade P. Simons K. Rotin D. J. Cell Biol. 2000; 149: 1473-1484Crossref PubMed Scopus (123) Google Scholar, 41Suzuki C. Murakami G. Fukuchi M. Shimanuki T. Shikauchi Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; 277: 39919-39925Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 42Wiesner S. Ogunjimi A.A. Wang H.R. Rotin D. Sicheri F. Wrana J.L. Forman-Kay J.D. Cell. 2007; 130: 651-662Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 54Morrione A. Plant P. Valentinis B. Staub O. Kumar S. Rotin D. Baserga R. J. Biol. Chem. 1999; 274: 24094-24099Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 55Ochoa W.F. Torrecillas A. Fita I. Verdaguer N. Corbalan-Garcia S. Gomez-Fernandez J.C. Biochemistry. 2003; 42: 8774-8779Crossref PubMed Scopus (72) Google Scholar, 56Grass I. Thiel S. Honing S. Haucke V. J. Biol. Chem. 2004; 279: 54872-54880Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 57Benes C.H. Wu N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The Rsp5 C2 domain binds to PI(3)P, however, replacement of the C2 domain with another PI(3)P binding domain, the Fab1 FYVE (Fab1, YOTB, Vac1, and EEA1) domain (58Kutateladze T.G. Ogburn K.D. Watson W.T. de Beer T. Emr S.D. Burd C.G. Overduin M. Mol. Cell. 1999; 3: 805-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) did not rescue the growth or MVE sorting phenotypes conferred by deletion of the C2 domain of Rsp5. 7A. Alder and L. Hicke, unpublished data. One explanation for" @default.
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• W2100524871 date "2008-12-01" @default.
• W2100524871 modified "2023-10-16" @default.
• W2100524871 title "A C-terminal Sequence in the Guanine Nucleotide Exchange Factor Sec7 Mediates Golgi Association and Interaction with the Rsp5 Ubiquitin Ligase" @default.
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• W2100524871 doi "https://doi.org/10.1074/jbc.m806023200" @default.
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