Purification and Cloning of a Brefeldin A-inhibited Guanine Nucleotide-exchange Protein for ADP-ribosylation Factors

Abstract

Activation of ADP-ribosylation factors (ARFs), ∼20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ∼200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ∼50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ∼209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ∼74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ∼202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 andBIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5′-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ∼50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ∼200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport. Activation of ADP-ribosylation factors (ARFs), ∼20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ∼200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ∼50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ∼209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ∼74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ∼202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 andBIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5′-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ∼50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ∼200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport. ADP-ribosylation factors (ARFs) 1The abbreviations used are: ARF(s), ADP-ribosylation factor(s); GEP, guanine nucleotide-exchange protein; BFA, brefeldin A; GTPγS, guanosine 5′-3-O-(thio)triphosphate PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); AEBSF, 4-(2-aminoethyl)butylsulfonyl fluoride; PH domain, pleckstrin homology domainare ∼20-kDa guanine nucleotide-binding proteins, initially identified by their ability to enhance cholera toxin-catalyzed ADP-ribosylation of the GTP-binding protein Gαs (1Kahn R.A. Gilman A.G. J. Biol. Chem. 1984; 259: 6228-6234Abstract Full Text PDF PubMed Google Scholar). ARFs are ubiquitous in eukaryotic cells from Giardia to human and are known to play an important role in intracellular vesicular trafficking (for review, see Ref. 2Moss J. Vaughan M. J. Biol. Chem. 1995; 270: 12327-12330Crossref PubMed Scopus (272) Google Scholar), as well as to activate phospholipase D (3Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar, 4Cockcroft S. Thomas G.M. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Truong O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar). Six mammalian ARFs, identified by cDNA cloning, are grouped into three classes based on size, amino acid sequence, phylogenetic analysis, and gene structure: ARFs 1–3 in class I, ARFs 4 and 5 in class II, and ARF6 in class III (5Tsuchiya M. Price S.R. Tsai S.-C. Moss J. Vaughan M. J. Biol. Chem. 1991; 266: 2772-2777Abstract Full Text PDF PubMed Google Scholar, 6Welsh C.F. Moss J. Vaughan M. Mol. Cell. Biochem. 1994; 138: 157-166Crossref PubMed Scopus (46) Google Scholar). Like other GTPases that regulate many kinds of intracellular processes, ARFs are active and associate with membranes when GTP is bound, whereas inactive ARF·GDP is cytosolic. Replacement of GDP by GTP is accelerated by ARF GEPs or guanine nucleotide-exchange proteins, several of which have been identified (7Moss J. Vaughan M. J. Biol. Chem. 1998; 273: 21431-21434Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). These include Gea1 and Gea2 from yeast (8Peyroche A. Paris S. Jackson C.L. Nature. 1996; 384: 479-481Crossref PubMed Scopus (236) Google Scholar), mammalian B2-1 or cytohesin-1 (9Liu L. Pohajdak B. Biochim. Biophys. Acta. 1992; 1132: 75-78Crossref PubMed Scopus (64) Google Scholar) and cytohesin-2 or ARNO (10Chardin P. Paris S. Antonny B. Robineau S. Béraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar), which are 83% identical in amino acid sequence, and GRP1 (general receptor forphosphoinositides), a third member of the cytohesin group (11Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (372) Google Scholar). All ARF GEPs of known structure contain Sec7 domains (8Peyroche A. Paris S. Jackson C.L. Nature. 1996; 384: 479-481Crossref PubMed Scopus (236) Google Scholar, 9Liu L. Pohajdak B. Biochim. Biophys. Acta. 1992; 1132: 75-78Crossref PubMed Scopus (64) Google Scholar, 10Chardin P. Paris S. Antonny B. Robineau S. Béraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar, 11Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (372) Google Scholar, 12Telemenakis I. Benseler F. Stenius K. Südhof T.C. Brose N. Eur. J. Cell. Biol. 1997; 74: 143-149PubMed Google Scholar, 13Franco M. Boretto J. Robineau S. Monier S. Goud B. Chardin P. Chavrier P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9926-9931Crossref PubMed Scopus (85) Google Scholar). Sec7 was identified in a group of conditionally lethal yeast mutants as a gene involved in Golgi vesicular trafficking and secretion (14Achstetter T. Franzusoff A. Field C. Schekman R. J. Biol. Chem. 1988; 263: 11711-11717Abstract Full Text PDF PubMed Google Scholar). The Sec7 gene product is a ∼230-kDa phosphoprotein that moves between membrane and cytosolic fractions (15Franzusoff A. Schekman R. EMBO J. 1989; 8: 2695-2702Crossref PubMed Scopus (170) Google Scholar). Its Sec7 domain was demonstrated relatively recently to function as a brefeldin A (BFA)-inhibited ARF GEP (16Sata M. Donaldson J.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4204-4208Crossref PubMed Scopus (78) Google Scholar). BFA is a fungal fatty acid metabolite with a monocyclic lactone ring that blocks protein secretion reversibly and causes apparent collapse of Golgi membranes into the endoplasmic reticulum (17Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar, 18Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 10: 801-813Abstract Full Text PDF Scopus (1315) Google Scholar). These effects result from BFA inhibition of GEP-catalyzed ARF activation (GTP binding) (19Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (596) Google Scholar, 20Helms J.B. Rothman J.E. Nature. 1992; 360: 352-354Crossref PubMed Scopus (585) Google Scholar). The two major types of ARF GEPs differ in size and susceptibility to inhibition by BFA. Their Sec7 domains contain the determinants of GEP activity as well as its BFA sensitivity. The larger ∼200-kDa GEPs (e.g. Sec7, Gea1, and Gea2 from yeast) are inhibited by BFA, whereas those of the cytohesin family are BFA-insensitive, ∼50-kDa proteins. Morinaga et al. (21Morinaga N. Tsai S.-C. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12856-12860Crossref PubMed Scopus (134) Google Scholar, 22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar) reported the purification and cloning of a BFA-inhibited ARF GEP from bovine brain, which was referred to as p200. We describe here the cloning from a human brain library of two GEP cDNAs. One, with a deduced amino acid sequence (1,849 residues) 99.5% identical to that of p200, probably represents the human form of p200. The other, encoding a 202-kDa protein 74% identical in sequence, is a new BFA-inhibited human GEP. We propose that because of their BFA sensitivity and size relative to the cytohesins, these mammalian ARF GEPs be named BIG1 (forBFA-inhibited GEP) and BIG2, respectively. [α-32P]dCTP, [γ-32P]dATP, and [35S]GTPγS were purchased from NEN Life Science Products. All restriction enzymes andTaq polymerase used to prepare BIG1-In and BIG2-In were purchased from Roche Molecular Biochemicals (formerly Boehringer Mannheim). Sources of other materials are noted below or have been reported (22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar). All plasmids were purified using Qiagen Plasmid Midi kits (Qiagen, Chatsworth, CA). An Applied Biosystems Inc. 373 DNA sequenator was used for of of and 8 of were used for of for for and for 4 in of for a human cDNA library were by with bovine BIG1 cDNA (22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar) as and in and was by with of in of the were with and for and and for Two were with The contained of the BIG2 named are relative to A of to The other contained of BIG1 One with contained of BIG1 and with contained of BIG2. The sequence between and was by a for the of for for and for were and in with of human cDNA as A of this was used as in a of for for and for with and The product was in to of the a BIG2. The sequence between and in BIG1 was by an for the of for for and for were in and in A of this product was used as in a of for for and for with and as The product was in of the of the BIG1 cDNA was by a of cDNA using DNA polymerase and a cDNA which the DNA and two with and in were and the of with and was used for of for for and for of this was used as in the with and for of for for and for The which was purified using a was in and of these cDNAs a BIG1 of BIG2 into the restriction for and were respectively, the and the of BIG2. the the was and BIG2 with and of as in a with 5 of polymerase for of for for and for The product was into The DNA from the purified in and of was to which had been with the and named and BIG2-In was the DNA with and and of polymerase for the of for for and for The product was purified using a and in of of product and of were used as in the of for for and for 4 with DNA and The which was purified using a was into The DNA was with of in The DNA and the which had been with were and an the were BIG2 and which an and the of BIG2 with DNA polymerase and of as in a of for of for for and for 4 The product was purified using a and into and of were used as for of with and in a of to The product was into based on with and A DNA from with and was to which had been with and the BIG2 the from with of was to which had been with and The DNA was named and was to the with the His6 sequence its A and of DNA were with cells in of for 5 a of the was to Sf9 cells in of by for 5 the was Sf9 cells were to of and of 5 cells were by in of and containing 8 of AEBSF, 5 of 5 of and 5 of on for and by and was from by The was with of acid 4 the was with of 5 and in and protein was with and in the of and The protein was 5 AEBSF, and 4 and with a human were to the was used for DNA a product in the of BIG2 and a product to in the of BIG1 were with using a DNA Molecular membranes were with and for and for and and for with of was with using of Molecular of this or other was used in of were in and for and for was The containing DNA from human cells cells, and cells, was purchased from the for DNA polymerase was used to prepare DNA for the BIG1 was with of DNA as the of the and of the for of for for and for An from BIG2 was from of human DNA with and in a of for of for for and for The product was to the of the in the and into DNA was used as in with and of the for of for for and for GEP of [35S]GTPγS binding to ARF was using a purified ARF ARFs and was from bovine brain (21Morinaga N. Tsai S.-C. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12856-12860Crossref PubMed Scopus (134) Google Scholar). which were for contained 4 of bovine and of with ARF and GEP as were by to by with of containing and were in for are of from BIG2 was initially purified as of a of from bovine brain. of by proteins of (p200) and BFA-inhibited ARF GEP activity (22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar). of from were used as the for cloning the bovine cDNA for of human BIG1 and BIG2 cDNAs is The cDNA for human BIG2 encodes a protein of amino with a of human and bovine BIG1 are larger with amino and of of by of the that had been purified from bovine brain (22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar) are in the deduced amino acid sequence of BIG2 are amino acids), a in the human is in the and in 8 amino acids), an in the bovine sequence is in the are and are from the Sec7 acid sequence of from is the deduced sequence from the human brain BIG2 and 8 are those amino that differ in the from purified bovine brain BIG2 (22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar). in a new is the deduced sequence from the human brain BIG2 and 8 are those amino that differ in the from purified bovine brain BIG2 (22Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar). The deduced amino acid sequence of human BIG2 is with those of human and bovine BIG1 in BIG2, which is 74% identical to human is identical in the amino of the Sec7 domain The BIG2 Sec7 domain is identical to the Sec7 domains of the BFA-insensitive and GRP1 are to those from yeast Sec7 and and identical to the Sec7 sequence from yeast Gea1 of the Sec7 domain, BIG1 and BIG2 have other of for to the yeast Sec7 of deduced amino acid sequences of Sec7 domains of ARF GEPs. The amino acid sequences are as in for (14Achstetter T. Franzusoff A. Field C. Schekman R. J. Biol. Chem. 1988; 263: 11711-11717Abstract Full Text PDF PubMed Google Scholar), I. Benseler F. Stenius K. Südhof T.C. Brose N. Eur. J. Cell. Biol. 1997; 74: 143-149PubMed Google Scholar), (8Peyroche A. Paris S. Jackson C.L. Nature. 1996; 384: 479-481Crossref PubMed Scopus (236) Google Scholar), (9Liu L. Pohajdak B. Biochim. Biophys. Acta. 1992; 1132: 75-78Crossref PubMed Scopus (64) Google Scholar), (10Chardin P. Paris S. Antonny B. Robineau S. Béraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar), and (11Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (372) Google Scholar). were by and by and of amino acid sequences of Sec7 domains of proteins with ARF GEP are and below the G. were as for of for sequences are in the for in a new of amino acid sequences of and or the of protein with Sec7 The of identical amino is in of the and the with from to the of the Sec7 were to of with or for the in of the two proteins. were by The and the were to a to a of size size are and below the G. were as for of for sequences are in the for with from human tissues a with BIG1 a with the BIG2 by and as a of the of the BIG1 mRNA in and was more than it was in brain, or The of BIG2 mRNA were relatively in of those The BIG1 cDNA with a from 8 not and with a of the size in the containing DNA The BIG2 with a from which was in the human DNA not with DNA from other chromosomes was the GEP activity of BIG2, it was synthesized as a His6 fusion protein in Sf9 purification on acid a protein to a of was on the purified BIG2 accelerated binding by in a concentration-dependent manner The of binding was for the of binding was to by 5 BIG2 did not these not of BIG2 on [35S]GTPγS binding by was with in for the without or with of BIG2. are the of from smaller than are not was BFA a of inhibited BIG2 activity and inhibition B17, a structural analog of BFA which not with Golgi did not BIG2 activity of BFA and on BIG2 of binding by activity relative to that in the of containing of and of BIG2 were without or with of BFA or for are of from activity relative to that in the of in a new containing of and of BIG2 were without or with of BFA or for are of from in BIG2 was in binding by and It activated the native ARF ARF1 and ARF3) more effectively than it did any of the recombinant nonmyristoylated of BIG2 on [35S]GTPγS binding by native ARF and recombinant class I, class II, and class III of native or with without or with of BIG2 were for are of from in a new of native or with without or with of BIG2 were for are of from BIG1 and BIG2, when initially identified as of a that was purified on the of its BFA-inhibited GEP were referred to as and (21Morinaga N. Tsai S.-C. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12856-12860Crossref PubMed Scopus (134) Google Scholar). are larger molecules than the ∼50-kDa BFA-insensitive We that be and BIG1 and BIG2. A cDNA for the protein, which been from a human brain is of from the bovine protein (21Morinaga N. Tsai S.-C. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12856-12860Crossref PubMed Scopus (134) Google Scholar) are identical of a of amino to that deduced from the human BIG2 in the of BIG1 and BIG2 a of with the that be of a of the GEP family the Sec7 domain a of the are other of the structure which its GEP activity (16Sata M. Donaldson J.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4204-4208Crossref PubMed Scopus (78) Google G. E. N. Moss J. Vaughan M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). are several of the Sec7 domains with sequences that are identical in BIG1 and BIG2 and have which to be The of other proteins of the GEP family (e.g. to function in different with a different of proteins and other to these proteins have other The of ARF GEPs in the and the or known ARFs (7Moss J. Vaughan M. J. Biol. Chem. 1998; 273: 21431-21434Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). are to the of and of the in which function. It that the GEPs in a major to a of ARF in cells a The of in BIG1 and BIG2 are the Sec7 which in the yeast Sec7 protein are for GEP activity and its BFA inhibition (16Sata M. Donaldson J.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4204-4208Crossref PubMed Scopus (78) Google Scholar). the BFA-insensitive GEP of ARNO (10Chardin P. Paris S. Antonny B. Robineau S. Béraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar) and cytohesin-1 E. Tsai S.-C. R. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) are of their Sec7 than amino in the Sec7 of which are identical in the BFA-sensitive and the for BFA inhibition should be the of cytohesin-1 and its Sec7 domain with a of proteins, it was that determinants of the of which is more than that of its Sec7 domain, in the G. E. N. Moss J. Vaughan M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). to be for the other ∼50-kDa BFA-insensitive ARNO (10Chardin P. Paris S. Antonny B. Robineau S. Béraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar) and GRP1 (11Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (372) Google Scholar). or not it to the ∼200-kDa BFA-inhibited GEPs to be The two families of GEPs differ in the of pleckstrin homology domains in the cytohesin-1 PH which in and are in nucleotide-exchange proteins for many GTPases and other molecules Science. 1997; 275: PubMed Scopus Google Scholar). cytohesin-1 and ARNO their PH S. Béraud-Dufour S. Robineau S. J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; PubMed Scopus Google Scholar), GRP1 a for J.K. L.C. Holik J.J. C. S. Czech M.P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the activity of the GEP because it the protein with the ARF a membrane S. Béraud-Dufour S. Robineau S. J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; PubMed Scopus Google Scholar). PH domains have not been in the larger BFA-sensitive their activity is by the A. I. P. P. J.S. G. R. T.C. A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) that activated ARF binding of and to Golgi it is to that an ARF GEP be of a containing those and other or molecules involved in a that is important for structure and function in vesicular trafficking It was that which is bound by the PH domain of the is of to the A is in a sequence the ARF of binding was not on its activation of phospholipase D or of proteins to the membrane et al. A. I. P. P. J.S. G. R. T.C. A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) that it from an in in Golgi BIG1 and BIG2 were purified as of a protein of purification from bovine brain on and by the BFA-inhibited GEP activity with a (21Morinaga N. Tsai S.-C. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12856-12860Crossref PubMed Scopus (134) Google Scholar). It was in a relatively from a of the of The active fractions contained several proteins that were by and the of which were to with from the and BFA-inhibited GEP activity (21Morinaga N. Tsai S.-C. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12856-12860Crossref PubMed Scopus (134) Google Scholar), as did not Although the other of the to be to be of in the regulation of vesicular transport. It of not been that the two in in the structure because it that the purified was a of from a of the of other it to that BIG1 and BIG2 be of different but with and probably in different intracellular in different from the of a that its proteins be is to the between the two gene their and their with other and of the We for