ADP-ribosylation factors (ARFs) and ARF-like 1 (ARL1) Have Both Specific and Shared Effectors
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m102359200
ISSN1083-351X
AutoresHillary Van Valkenburgh, Jack F. Shern, J. Daniel Sharer, Xinjun Zhu, Richard Kahn,
Tópico(s)PARP inhibition in cancer therapy
ResumoDespite the 40–60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1 (1Zhang C.J. Rosenwald A.G. Willingham M.C. Skuntz S. Clark J. Kahn R.A. J. Cell Biol. 1994; 124: 289-300Crossref PubMed Scopus (151) Google Scholar). Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously. Despite the 40–60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1 (1Zhang C.J. Rosenwald A.G. Willingham M.C. Skuntz S. Clark J. Kahn R.A. J. Cell Biol. 1994; 124: 289-300Crossref PubMed Scopus (151) Google Scholar). Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously. ADP-ribosylation factor(s) ARF-like binder of ARL2 δ subunit of cGMP phosphodiesterase 6 normal rat kidney guanosine 5′-3-O-(thio)triphosphate bovine serum albumin GTPase-activating protein short coiled-coil human retinal gene 4-morpholinepropanesulfonic acid base pairs hemagglutinin untranslated region guanine nucleotide exchange factor ADP-ribosylation factors (ARFs)1 are highly conserved, ubiquitous, 21-kDa GTP-binding proteins with roles in multiple steps of membrane traffic and other cellular processes (for review, see Refs.2Boman A.L. Kahn R.A. Trends Biochem. Sci. 1995; 20: 147-150Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 3Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2072) Google Scholar, 4Cullen P.J. Venkateswarlu K. Biochem. Soc. 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The ARF-like (ARL) proteins are 40–60% identical to each other or to any ARF and are essentially devoid of the activities described for ARFs (12Stearns T. Kahn R.A. Botstein D. Hoyt M.A. Mol. Cell. Biol. 1990; 10: 6690-6699Crossref PubMed Scopus (199) Google Scholar, 15Tamkun J.W. Kahn R.A. Kissinger M. Brizuela B.J. Rulka C. Scott M.P. Kennison J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3120-3124Crossref PubMed Scopus (124) Google Scholar,16Hong J.X. Lee F.J. Patton W.A. Lin C.Y. Moss J. Vaughan M. J. Biol. Chem. 1998; 273: 15872-15876Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). A third group of proteins, including yeast SAR1 and CIN4, is included in the ARF family although the proteins share only 25–35% sequence identity and have clearly distinct activities in cells (5Roth M.G. Hall A. GTPases. Oxford University Press, New York2000: 176-197Google Scholar,17Botstein D. Segev N. Stearns T. Hoyt M.A. Holden J. Kahn R.A. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 629-636Crossref PubMed Google Scholar). More than 10 ARLs have been identified in humans, and three in S. cerevisiae (15Tamkun J.W. Kahn R.A. Kissinger M. Brizuela B.J. Rulka C. Scott M.P. Kennison J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3120-3124Crossref PubMed Scopus (124) Google Scholar, 18Clark J. Moore L. Krasinskas A. Way J. Battey J. Tamkun J. Kahn R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8952-8956Crossref PubMed Scopus (111) Google Scholar, 19Schurmann A. Breiner M. Becker W. Huppertz C. Kainulainen H. Kentrup H. Joost H.G. J. Biol. Chem. 1994; 269: 15683-15688Abstract Full Text PDF PubMed Google Scholar, 20Zhang G.F. Patton W.A. Lee F.J. Liyanage M. Han J.S. Rhee S.G. Moss J. Vaughan M. J. Biol. Chem. 1995; 270: 21-24Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Lowe S.L. Wong S.H. Hong W. J. Cell Sci. 1996; 109: 209-220Crossref PubMed Google Scholar). Although ARFs have been purified repeatedly by laboratories using different biochemical assays, no ARL has ever been purified or cloned based on an activity. The lack of ARL activity in ARF assays has led to the conclusion that ARLs have distinct biochemical activities and thus cellular functions, despite their similarity in sequence and structure (15Tamkun J.W. Kahn R.A. Kissinger M. Brizuela B.J. Rulka C. Scott M.P. Kennison J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3120-3124Crossref PubMed Scopus (124) Google Scholar). 2J. C. Amor, X. Zhu, J. Horton, X. Cheng, D. Ringe, and R. A. Kahn, manuscript in preparation. The lack of functional overlap between ARFs and ARLs is evident from the findings that ARLs in yeast cannot rescue the synthetic lethality of thearf1 − arf2 −mutations (12Stearns T. Kahn R.A. Botstein D. Hoyt M.A. Mol. Cell. Biol. 1990; 10: 6690-6699Crossref PubMed Scopus (199) Google Scholar), whereas any of the six mammalian ARFs (13Kahn R.A. Kern F.G. Clark J. Gelmann E.P. Rulka C. J. Biol. Chem. 1991; 266: 2606-2614Abstract Full Text PDF PubMed Google Scholar, 23Lee F.J. Moss J. Vaughan M. J. Biol. Chem. 1992; 267: 24441-24445Abstract Full Text PDF PubMed Google Scholar) or ARFs from other organisms (e.g. Drosophila (24Murtagh J.J. Lee F.J. Deak P. Hall L.M. Monaco L. Lee C.M. Stevens L.A. Moss J. Vaughan M. Biochemistry. 1993; 32: 6011-6018Crossref PubMed Scopus (25) Google Scholar), orGiardia (23Lee F.J. Moss J. Vaughan M. J. Biol. Chem. 1992; 267: 24441-24445Abstract Full Text PDF PubMed Google Scholar)) can suppress the lethality ofarf1 − arf2 − in yeast. Similarly, deletion of the Drosophila arl1 gene causes zygotic lethality despite the presence of the full complement of ARF genes in flies (15Tamkun J.W. Kahn R.A. Kissinger M. Brizuela B.J. Rulka C. Scott M.P. Kennison J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3120-3124Crossref PubMed Scopus (124) Google Scholar). In contrast to the many activities and functions assigned to ARFs, none has yet been assigned to any ARL from any species. The number of binding partners for mammalian ARF proteins has increased dramatically in recent years in large part because of the use of two-hybrid screens that have identified seven new ARF effectors: Arfaptin1 (25Kanoh H. Williger B.T. Exton J.H. J. Biol. Chem. 1997; 272: 5421-5429Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), Arfaptin2/POR1 (independently isolated by two groups (25Kanoh H. Williger B.T. Exton J.H. J. Biol. Chem. 1997; 272: 5421-5429Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 26Van Aelst L. Joneson T. Bar-Sagi D. EMBO J. 1996; 15: 3778-3786Crossref PubMed Scopus (144) Google Scholar) and named Arfaptin 2 and POR1, respectively), MKLP1 (27Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar), Arfophilin (28Shin O.H. Ross A.H. Mihai I. Exton J.H. J. Biol. Chem. 1999; 274: 36609-36615Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and the three GGAs (29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google Scholar, 30Hirst J. Lui W.W. Bright N.A. Totty N. Seaman M.N. Robinson M.S. J. Cell Biol. 2000; 149: 67-80Crossref PubMed Scopus (272) Google Scholar, 31Dell'Angelica E.C. Puertollano R. Mullins C. Aguilar R.C. Vargas J.D. Hartnell L.M. Bonifacino J.S. J. Cell Biol. 2000; 149: 81-94Crossref PubMed Scopus (326) Google Scholar). The use of carboxyl-terminal fusion proteins and dominant activated ARF mutants has enhanced the ability to select for binding partners that interact preferentially with the activated form of the GTPase (27Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google Scholar, 32Zhu X. Boman A.L. Kuai J. Cieplak W. Kahn R.A. J. Biol. Chem. 2000; 275: 13465-13475Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Two proteins, "binder of ARL2" (BART (33Sharer J.D. Kahn R.A. J. Biol. Chem. 1999; 274: 27553-27561Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar)) and the δ subunit of cGMP phosphodiesterase 6 (PDEδ (34Linari M. Hanzal-Bayer M. Becker J. FEBS Lett. 1999; 458: 55-59Crossref PubMed Scopus (80) Google Scholar)), have been cloned based on their GTP-dependent interaction with either ARL2 or ARL3, respectively. To date, however, no clear biological significance has been attributed to these interactions. Role(s) for ARF in Golgi functions were evident when expression of the persistently activated mutant allele [Q71L]ARF1 in cells led to expansion and vesiculation of the Golgi compartment (1Zhang C.J. Rosenwald A.G. Willingham M.C. Skuntz S. Clark J. Kahn R.A. J. Cell Biol. 1994; 124: 289-300Crossref PubMed Scopus (151) Google Scholar). Because ARL1 was reported also to bind Golgi membranes in intact cells, we used the same procedures to test for a functional role of ARL1 at the Golgi. We were surprised by the similarities between effects of activated ARF1 and ARL1 on Golgi morphology, and this led us to reexamine the extent of functional overlap between ARF and ARL proteins. Every experiment reported herein was repeated at least twice and in most cases additional times with essentially the same results. Growth media were prepared, and maintenance of yeast strains was performed as described in Sherman et al.(35Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1974Google Scholar). Transformation of yeast was performed using the method of Schiestl and Gietz (36Schiestl R.H. Gietz R.D. Curr Genet. 1989; 16: 339-346Crossref PubMed Scopus (1776) Google Scholar). Plasmids were rescued from yeast as described (37Hoffman C.S. Winston F. Gene ( Amst. ). 1987; 57: 267-272Crossref PubMed Scopus (2057) Google Scholar) followed by further purification on Qiagen minipreparation columns. DNA was transformed into E. coli strain DH5α prior to plasmid preparation. Normal rat kidney (NRK) cells were obtained from American Type Culture Collection (ATCC; Rockville, MD) and grown and passaged in RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Life Technologies, Inc.) at 37 °C in a humidified atmosphere containing 10% CO2. Yeast strains Y190 (MATa gal4 gal80 his3 trp1–901 ade2–101 ura3–52 leu2–3, -112 URA3::GAL→lacZ) and Y187 (MATαgal4 gal80 his3 trp1–901 ade2–101 ura3–52 leu2–3,-112 met- URA3::GAL→lacZ), plasmids pAS2 and pACT2, and the human B cell library in pACT were the gifts of Steven J. Elledge (Baylor University). This system utilizes the GAL4 binding and activation domains and allows for two independent read-outs for a positive interaction in the two-hybrid system: histidine auxotrophy and β-galactosidase expression (38Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar). The human fetal brain cDNA library, in pACT2, was purchased fromCLONTECH. Plasmid pBG4D was a gift from Rob Brazas and allowed the expression of proteins fused through their carboxyl termini to the GAL4 binding domain. The open reading frame of each ARL was cloned in-frame into pBG4D via BamHI and NotI sites. Mutations were generated using Stratagene's QuikChange site-directed mutagenesis kit. All DNA generated by polymerase chain reaction was sequenced both to confirm the presence of designed mutations and prevent the introduction of others. Screening of human cDNA libraries was performed as described previously (38Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar) with the modifications described in Boman et al. (27Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google Scholar). Briefly, the dominant activating mutations in each human ARL protein were used as bait to screen human fetal brain or B cell libraries in Y190 by selection for growth on selective plates containing 25 mm 3-aminotriazole (3-AT). Positives were then assayed for β-galactosidase activity, using the nitrocellulose filter binding assay of Breeden and Lasmyth (39Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar). Filters were incubated at 30 °C for up to 3 h. A strong interaction was defined as the development of a dark blue color within 15 min; a weak interaction required the full 3 h for development of a pale blue color. Further tests for specificity of interactions included: 1) counterselection for the loss of the ARL plasmid on cycloheximide plates resulted in loss of activity in the β-galactosidase assay; 2) cells carrying each potential positive were mated to unrelated partners (we used CDK2, lamin, and p53, each fused to the GAL4 activation domain) and assayed for β-galactosidase activity; and 3) rescued library plasmids yielded growth on 3-AT plates and β-galactosidase activity after transformation into the original ARL-bearing yeast strain. In most cases, positives were counterscreened against the wild type protein, to help identify those proteins that interacted preferentially with the GTP-bound form of the GTPase. TableI lists each of the plasmids used in the studies described below.Table IPlasmids used in this studyPlasmid nameInsertVectorRef.pAB155ARF3-BDpBG4D27Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpAB157[Q71L]ARF3-BDpBG4D27Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpJCH1–11BD-[Q71L]ARF1pAS127Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpJCH3–2BD-[Q71L]ARF3pAS127Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpJCH2–14BD-[Q71L]ARF4pAS127Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpJCH5–12BD-[Q71L]ARF5pAS127Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpJCH4–13BD-[Q71L]ARF6pAS127Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google Scholar, 29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpHV330ARL1-BDpBG4DThis paperpHV628[Q71L]ARL1-BDpBG4DThis paperpJS100–1ARL2-BDpBG4DThis paperpJS106[Q70L]ARL2-BDpBG4DThis paperpJS104ARL3-BDpBG4DThis paperpJS103[Q71L]ARL3-BDpBG4DThis paperpAB169AD-GGA1(145–639)pACT29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpAB195AD-GGA2(131–613)pACT29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpAB179AD-GGA3(80–690)pACT29Boman A.L. Zhang C. Zhu X. Kahn R.A. Mol. Biol. Cell. 2000; 11: 1241-1255Crossref PubMed Scopus (225) Google ScholarpAB138AD-[E112D]LTApACT69Zhu X. Kim E. Boman A.L. Hodel A. Cieplak W. Kahn R.A. Biochemistry. 2001; 40: 4560-4568Crossref PubMed Scopus (14) Google ScholarpAB199AD-MKLP1 (663–960)pACT27Boman A.L. Kuai J. Zhu X. Chen J. Kuriyama R. Kahn R.A. Cell Motil. Cytoskeleton. 1999; 44: 119-132Crossref PubMed Scopus (58) Google ScholarpGAD-POR1ΔNAD-POR1 (79–341)pACT226Van Aelst L. Joneson T. Bar-Sagi D. EMBO J. 1996; 15: 3778-3786Crossref PubMed Scopus (144) Google ScholarpHV839AD-Arfaptin2/POR1pACTThis paperp2QC-36AD-BARTpACT2This paperpHV842AD-Golgin-245(2025–2083)pACTThis paperpJSQB86AD-HRG4pACTThis paperpHV840AD-MKLP1(456–960)pACTThis paperpJS2QC60BAD-PDEδpACT2This paperpHV841AD-SCOCOpACTThis paperpHV883AD[Y2032A]Golgin-245(2025–2083)pACTThis paperpHV631ARL1-(His)6pET20This paperpHV862SCOCO-(His)6pET20This paperpHV859SCOCOpET3CThis paperpBART-(His)6BART-(His)6pET3C33Sharer J.D. Kahn R.A. J. Biol. Chem. 1999; 274: 27553-27561Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarpHV870(His)6-Golgin-245(2025–2083)pET14This paperpHV877[Δ17]ARL1-(His)6pET20This paperpHV627ARL1-mycpSS2–2This paperpHV620[Q71L]ARL1-mycpSS2–2This paperpHV82050-bp 5′-UTR-ARL1-HApSS2–2/NEOThis paperpHV81050-bp 5′-UTR [Q71L]ARL1-HApSS2–2/NEOThis paperpHV873Golgin-245(2025–2083)-myc-(His)6pCDNA3.1 Myc-HisThis paperpHV872SCOCO-myc-(His)6pCDNA3.1 Myc-HisThis paperpHV885[Y2032A]Golgin-245(2025–2083)-myc-(His)6pCDNA3.1 Myc-HisThis paperColumns indicate the name of the plasmid, the insert, parental plasmid, and source. When a fragment of an open reading frame is used, the residues included in the construct are indicated in parentheses after the name of the insert. AD, GAL4 activation domain; BD, GAL4 binding domain. Open table in a new tab Columns indicate the name of the plasmid, the insert, parental plasmid, and source. When a fragment of an open reading frame is used, the residues included in the construct are indicated in parentheses after the name of the insert. AD, GAL4 activation domain; BD, GAL4 binding domain. The open reading frames of human ARL1 and [Δ17]ARL1 were subcloned into pET20b (Novagen) at the NdeI and NotI sites (Table I). The open reading frames of SCOCO and the GRIP domain of Golgin-245 (amino acids 2025–2083) were cloned into pET20b (via NdeI and NotI restriction sites) and pET14b (via NdeI and BamHI restriction sites), respectively (Table I). The expression of (His)6-tagged protein was induced for 3 h in mid-log phase BL21(DE3) cells with 1 mm isopropyl β-d-thiogalactopyranoside at 37 °C. Cells were lysed with a French pressure cell, and the recombinant protein was purified using a 1-ml HiTrap chelating (Amersham Pharmacia Biotech) column charged with nickel chloride and developed with a 0–500 mm imidazole gradient according to the manufacturer's instructions. Fractions that contained (His)6-tagged protein, as determined by SDS-polyacrylamide gel electrophoresis, were pooled, and the buffer was exchanged into 25 mm Tris, pH 7.4, 100 mm NaCl, 2 mmMgCl2, and 1 mm dithiothreitol. Human ARL2, ARL3, and ARF1 cDNA were all cloned into pET3C via NdeI and BamHI restriction sites (Table I). They were each expressed and purified as described previously (18Clark J. Moore L. Krasinskas A. Way J. Battey J. Tamkun J. Kahn R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8952-8956Crossref PubMed Scopus (111) Google Scholar, 33Sharer J.D. Kahn R.A. J. Biol. Chem. 1999; 274: 27553-27561Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). POR1 fused to the maltose-binding protein (a gift from Linda Van Aelst) was purified as described (26Van Aelst L. Joneson T. Bar-Sagi D. EMBO J. 1996; 15: 3778-3786Crossref PubMed Scopus (144) Google Scholar). The GTPγS binding assay was performed at 30 °C in 20 mm HEPES, pH 7.4, 100 mm NaCl, 1 mm dithiothreitol, 1 mmEDTA, 0.5 mm MgCl2, 50 μg/ml BSA, and 10 μm [γ-35S]GTPγS (1,000 cpm/pmol), as described previously (40Kahn R.A. Methods Enzymol. 1991; 195: 233-242Crossref PubMed Scopus (19) Google Scholar). ARL1 GAP activity was assayed by a modification of the method of Randazzo and Kahn (41Randazzo P.A. Kahn R.A. J. Biol. Chem. 1994; 269: 10758-10763Abstract Full Text PDF PubMed Google Scholar) using 0.18 μmpurified, recombinant [Δ17]ARL1-(His)6 as the substrate with or without the addition of 1.8 μm GRIP domain of Golgin-245 or SCOCO. Purified, recombinant human ARF3 and ARF GAP (42Huber I. Cukierman E. Rotman M. Aoe T. Hsu V.W. Cassel D. J. Biol. Chem. 1998; 273: 24786-24791Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) served as positive controls for the assay. Direct interaction between human ARL1, 2, or 3 and BART, PDEδ, or HRG4 was assayed by gel overlay, as described in Sharer and Kahn (33Sharer J.D. Kahn R.A. J. Biol. Chem. 1999; 274: 27553-27561Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, SDS-solubilized protein lysates were prepared from BL21(DE3) cells containing either the PDEδ open reading frame in pET15b (the gift of Ahmed Zahraoui, Compartimentation et Dynamique Cellulaires, Institut Curie, Paris, France), HRG4 open reading frame in pET14b at NdeI and BamHI restriction sites, or an empty vector control (Table I). 25 μg of total protein was resolved on a 15% polyacrylamide gel before electrophoretic transfer to 0.2-μm nitrocellulose membrane (Bio-Rad). Proteins adsorbed on the filter were renatured in 10 mmMOPS, pH 7.1, 100 mm potassium acetate, 0.25% Tween 20, 5 mm magnesium acetate, 0.5% BSA, 5 mmdithiothreitol, and incubated with 2 μg of recombinant [Δ17]ARL1-(His)6, ARL2, or ARL3 prebound to 20 μCi of [α-32P]GTP. The filter was washed three times with binding buffer (20 mm MOPS, pH 7.1, 100 mmpotassium acetate, 0.1% Triton X-100, 5 mm magnesium acetate, 0.5% BSA, 50 μm GTP, 50 μm GDP, and 5 mm dithiothreitol), and specific binding was determined by phosphorimage analysis. 10 μm purified recombinant [Δ17]ARL1-(His)6 was loaded with either 100 μm GDP or GTPγS for 15 min at 30 °C in the presence of 20 mm HEPES, pH 7.4, 100 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 0.5 mm MgCl2, and 50 μg/ml BSA. SCOCO was covalently attached to Affi-Gel 15 beads according to the manufacturer's directions (Bio-Rad). SCOCO beads were washed twice in binding buffer containing 10 μm appropriate nucleotide, just prior to the addition of preloaded [Δ17]ARL1-(His)6. SCOCO beads and [Δ17]ARL1-GDP or -GTPγS were incubated with gentle rocking for 15 min at room temperature. The beads were washed twice with binding buffer, containing 20 μm appropriate nucleotide and then mixed with an equivalent amount of 2 × Laemmli sample buffer. Proteins were resolved on a 15% polyacrylamide gel, and the presence of [Δ17]ARL1-(His)6 was detected using polyclonal rabbit antiserum raised against ARL1 (R85722-3). NRK cells were fixed in 3.7% formaldehyde and permeabilized in 0.2% saponin with 10% goat serum, as described in Zhang et al. (1Zhang C.J. Rosenwald A.G. Willingham M.C. Skuntz S. Clark J. Kahn R.A. J. Cell Biol. 1994; 124: 289-300Crossref PubMed Scopus (151) Google Scholar). Cells were visualized either on an Olympus BX60 fluorescent microscope or a Bio-Rad 1024 laser scanning confocal microscope coupled to a Zeiss Axioskop. For confocal microscopy, a Z-series of images was collected with 5-μm steps and processed using IMAGE J software from NIH IMAGE. Recombinant SCOCO-(His)6 was purified as described above and used as antigen in rabbits, after conjugation to keyhole limpet hemocyanin through both the NH2 terminus (using glutaraldehyde) and the COOH terminus (using carbodiimide). Antibodies were affinity purified by sequential protein G-Sepharose and affinity chromatography. 6.5 mg of untagged, recombinant SCOCO was covalently attached to 1-ml Affi-Gel 15 beads, according to the manufacturer's directions. Serum from rabbit R97679 was enriched for immunoglobulins on a 1-ml protein G column (Amersham Pharmacia Biotech) and eluted with 0.1 m glycine HCl, pH, 2.7. The eluted antibodies were exchanged into 0.1 m MOPS, pH 7.2, and applied to the Affi-Gel 15-SCOCO column. The column was washed with 10 ml of phosphate-buffered saline containing 1 m NaCl. Antibodies were eluted with 10 ml of 0.1 m glycine-HCl, pH 2.4, collecting 1-ml fractions into 100 μl of 1 mTris-HCl, pH 9, to neutralize the glycine buffer. Fractions containing protein were pooled, and the buffer was exchanged for phosphate-buffered saline. COOH-terminal, myc-epitope tagged constructs of ARL1 or [Q71L]ARL1 were subcloned into pSS2-2 for expression in mammalian cells under regulation by the interferon-inducible Mx1 promoter, as described in Zhang et al. (Table I and Ref. 1Zhang C.J. Rosenwald A.G. Willingham M.C. Skuntz S. Clark J. Kahn R.A. J. Cell Biol. 1994; 124: 289-300Crossref PubMed Scopus (151) Google Scholar). Each of these or the parental vector was cotransfected with pSV2-neo (ATCC), at a 10:1 ratio of DNA, into NRK cells using Fugene 6 (Invitrogen) reagent, following the manufacturer's directions. 48 h after transfection, the cells were split and diluted into medium containing 400 μg/ml Geneticin disulfate (G418; Sigma). G418-resistant clones were isolated and later cloned by limited dilution. Cell lines carrying stably integrated plasmids directing expression of ARL1-myc or [Q71L]ARL1-myc with no ARL1 5′-untranslated sequence were NRK-HV1–9 and NRK-HV2–20, respectively. Protein expression was induced with 1,000 units/ml α, β-rat interferon (Lee Biomolecular, San Diego) and assayed by immunoblot analysis using monoclonal 9E10 (mouse α-myc) antibodies. When lysates were probed with myc antibodies in immunoblots, we consistently observed the interferon-dependent expression of a doublet in which the upper band corresponded to the predicted size of full-length ARL1 (21 kDa) and a second band migrating as a smaller fragment (≈17 kDa). Human ARL1 is myristoylated at its NH2 terminus (43Lee F.J. Huang C.F., Yu, W.L. Buu L.M. Lin C.Y. Huang M.C. Moss J. Vaug
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