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Human and Viral Golgi Anti-apoptotic Proteins (GAAPs) Oligomerize via Different Mechanisms and Monomeric GAAP Inhibits Apoptosis and Modulates Calcium*

  • Nuno Saraiva
    Footnotes
    Affiliations
    Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom

    Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom
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  • David L. Prole
    Footnotes
    Affiliations
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom
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  • Guia Carrara
    Affiliations
    Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom

    Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom
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  • Carlos Maluquer de Motes
    Affiliations
    Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom

    Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom
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  • Benjamin F. Johnson
    Affiliations
    Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom
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  • Bernadette Byrne
    Affiliations
    Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom
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  • Colin W. Taylor
    Affiliations
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom
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  • Geoffrey L. Smith
    Correspondence
    A Wellcome Trust principal research fellow. To whom correspondence should be addressed: Dept. of Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, UK. Tel.: 44-1223-336922; Fax: 44-1223-339067;
    Affiliations
    Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom

    Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom
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  • Author Footnotes
    * This work was supported by grants from the UK Medical Research Council (G0900224) and The Wellcome Trust (085295).
    This article contains supplemental Figs. S1 and S2.
    1 Supported by the Portuguese Foundation for Science and Technology.
    2 Supported in part by a Meres Research associateship from St. John's College (Cambridge, UK).
Open AccessPublished:March 18, 2013DOI:https://doi.org/10.1074/jbc.M112.414367
      Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca2+ content of intracellular stores, and regulate Ca2+ fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca2+-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca2+ stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

      Introduction

      The gene encoding Golgi anti-apoptotic protein (GAAP)
      The abbreviations used are: GAAP
      Golgi anti-apoptotic protein
      BI-1
      Bax inhibitor-1
      [Ca2+]i
      intracellular free Ca2+ concentration
      CMLV
      camelpox virus
      DFDNB
      1,5-difluoro-2,4-dinitrobenzene
      ER
      endoplasmic reticulum
      vGAAP
      viral GAAP
      hGAAP
      human GAAP
      TMBIM
      transmembrane BI-1-containing motif
      TMD
      transmembrane domain
      Tom
      tandem tomato
      VACV
      vaccinia virus.
      was initially identified in camelpox virus (CMLV) and some vaccinia virus (VACV) strains (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ). Deletion of the GAAP gene from the VACV genome had no effect on virus replication in cell culture but altered virus virulence in vivo (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ). Cells expressing viral GAAP (vGAAP) displayed resistance to apoptosis triggered by a wide range of intrinsic and extrinsic stimuli (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ).
      These viral proteins are closely related (73% amino acid identity) to the ubiquitously expressed human GAAP (hGAAP) (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ). hGAAP was identified as a possible housekeeping gene because it is essential for cell viability (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ) and from statistical analysis of microarrays (
      • Lee S.
      • Jo M.
      • Lee J.
      • Koh S.S.
      • Kim S.
      Identification of novel universal housekeeping genes by statistical analysis of microarray data.
      ). Furthermore, its mRNA levels are dysregulated in some human breast tumors, making it a putative oncogene and a possible target for anti-cancer therapy (
      • van ‘t Veer L.J.
      • Dai H.
      • van de Vijver M.J.
      • He Y.D.
      • Hart A.A.
      • Mao M.
      • Peterse H.L.
      • van der Kooy K.
      • Marton M.J.
      • Witteveen A.T.
      • Schreiber G.J.
      • Kerkhoven R.M.
      • Roberts C.
      • Linsley P.S.
      • Bernards R.
      • Friend S.H.
      Gene expression profiling predicts clinical outcome of breast cancer.
      ).
      GAAPs are highly conserved, and closely related proteins are predicted to be encoded by the genomes of fungi, plants, and animals (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ). All GAAPs, from evolutionarily diverse sources, have similar lengths and hydrophobicity profiles that point to important and conserved functions. Cells in which hGAAP was knocked down using siRNA died but were rescued by expression of vGAAP (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ), suggesting that the two GAAPs are functionally interchangeable.
      Phylogenetic analysis suggests that GAAPs are ancient within eukaryotes. Some members of the transmembrane Bax (Bcl-2-associated X protein) inhibitor-containing motif (TMBIM) family may have derived from a GAAP ancestor ∼2,000 million years ago (
      • Hu L.
      • Smith T.F.
      • Goldberger G.
      LFG: a candidate apoptosis regulatory gene family.
      ). Members of the TMBIM family include TMBIM1 or RESC (responsive to centrifugal force and shear stress gene 1 protein), TMBIM2 or LFG (lifeguard protein), TMBIM3 or GRINA (glutamate receptor ionotropic NMDA-associated protein), TMBIM4 or GAAP, TMBIM5 or GHITM (growth hormone-inducible transmembrane protein), and TMBIM6 or BI-1 (Bax inhibitor 1) (
      • Gubser C.
      • Bergamaschi D.
      • Hollinshead M.
      • Lu X.
      • van Kuppeveld F.J.
      • Smith G.L.
      A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
      ,
      • Hu L.
      • Smith T.F.
      • Goldberger G.
      LFG: a candidate apoptosis regulatory gene family.
      ,
      • Xu Q.
      • Reed J.C.
      Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast.
      ,
      • Rojas-Rivera D.
      • Armisén R.
      • Colombo A.
      • Martínez G.
      • Eguiguren A.L.
      • Díaz A.
      • Kiviluoto S.
      • Rodríguez D.
      • Patron M.
      • Rizzuto R.
      • Bultynck G.
      • Concha M.L.
      • Sierralta J.
      • Stutzin A.
      • Hetz C.
      TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.
      ,
      • Zhou J.
      • Zhu T.
      • Hu C.
      • Li H.
      • Chen G.
      • Xu G.
      • Wang S.
      • Zhou J.
      • Ma D.
      Comparative genomics and function analysis on BI1 family.
      ). All TMBIM proteins are predicted to have similar secondary structures, consisting of multiple transmembrane domains (TMDs) with short interconnecting loops, a charged C terminus, and a conserved UPF0005 motif with unknown function that stretches from the beginning of the third to the middle of the fourth TMD (
      • Hu L.
      • Smith T.F.
      • Goldberger G.
      LFG: a candidate apoptosis regulatory gene family.
      ,
      • Reimers K.
      • Choi C.Y.
      • Bucan V.
      • Vogt P.M.
      The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis.
      ). Recently, vGAAP, hGAAP, and BI-1 were shown to have a six TMD topology with cytosolic N and C termini and a likely re-entrant loop at the C terminus (
      • Carrara G.
      • Saraiva N.
      • Gubser C.
      • Johnson B.F.
      • Smith G.L.
      Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family.
      ).
      Similar to GAAP, BI-1 is a widely expressed and evolutionarily conserved protein that protects cells from many different intrinsic and extrinsic death stimuli (
      • Xu Q.
      • Reed J.C.
      Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast.
      ). It is the most studied member of the TMBIM family. BI-1 localizes primarily to endoplasmic reticulum (ER) membranes, where it causes pH-dependent Ca2+ leakage that reduces the ER Ca2+ content, and so the amount of Ca2+ released upon stimulation (
      • Xu C.
      • Xu W.
      • Palmer A.E.
      • Reed J.C.
      BI-1 regulates endoplasmic reticulum Ca2+ homeostasis downstream of Bcl-2 family proteins.
      ). Work with purified BI-1 reconstituted into liposomes suggests it has a Ca2+/H+ antiporter-like (
      • Ahn T.
      • Yun C.H.
      • Chae H.Z.
      • Kim H.R.
      • Chae H.J.
      Ca2+/H+ antiporter-like activity of human recombinant Bax inhibitor-1 reconstituted into liposomes.
      ) or Ca2+ channel activity (
      • Bultynck G.
      • Kiviluoto S.
      • Henke N.
      • Ivanova H.
      • Schneider L.
      • Rybalchenko V.
      • Luyten T.
      • Nuyts K.
      • De Borggraeve W.
      • Bezprozvanny I.
      • Parys J.B.
      • De Smedt H.
      • Missiaen L.
      • Methner A.
      The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.
      ). Moreover, two aspartic acid residues (Asp209 and Asp213) in BI-1 are essential for Ca2+ flux from the ER (
      • Bultynck G.
      • Kiviluoto S.
      • Henke N.
      • Ivanova H.
      • Schneider L.
      • Rybalchenko V.
      • Luyten T.
      • Nuyts K.
      • De Borggraeve W.
      • Bezprozvanny I.
      • Parys J.B.
      • De Smedt H.
      • Missiaen L.
      • Methner A.
      The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.
      ). Regulation of the Ca2+ content of the ER by BI-1 suggests a possible mechanism for its anti-apoptotic function.
      GAAPs localize primarily to Golgi membranes and, similar to BI-1, have a broad spectrum of anti-apoptotic activity. This suggests a possible link with intracellular Ca2+, which plays important roles in apoptosis triggered by different stimuli (
      • Orrenius S.
      • Zhivotovsky B.
      • Nicotera P.
      Regulation of cell death: the calcium-apoptosis link.
      ). This suggestion is also consistent with measurements of the Ca2+ content of organelles after overexpression or knockdown of hGAAP (
      • de Mattia F.
      • Gubser C.
      • van Dommelen M.M.
      • Visch H.J.
      • Distelmaier F.
      • Postigo A.
      • Luyten T.
      • Parys J.B.
      • de Smedt H.
      • Smith G.L.
      • Willems P.H.
      • van Kuppeveld F.J.
      Human Golgi antiapoptotic protein modulates intracellular calcium fluxes.
      ). Overexpression of hGAAP reduced the Ca2+ content of the Golgi and ER and the amount of Ca2+ released by an apoptotic stimulus (
      • de Mattia F.
      • Gubser C.
      • van Dommelen M.M.
      • Visch H.J.
      • Distelmaier F.
      • Postigo A.
      • Luyten T.
      • Parys J.B.
      • de Smedt H.
      • Smith G.L.
      • Willems P.H.
      • van Kuppeveld F.J.
      Human Golgi antiapoptotic protein modulates intracellular calcium fluxes.
      ). Therefore, the effects of GAAPs on Ca2+ signaling might explain their broad anti-apoptotic activity, although a causal link is yet to be established.
      The membrane topology of TMBIM proteins has been established (
      • Carrara G.
      • Saraiva N.
      • Gubser C.
      • Johnson B.F.
      • Smith G.L.
      Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family.
      ), but there are no further structural data. Most TMBIM proteins show multiple bands on SDS-PAGE, suggesting the existence of oligomers. However, neither the oligomeric state of TMBIM proteins in native membranes nor the determinants or functional consequences of oligomerization are known. To address these questions, the mechanisms of oligomerization of vGAAP and hGAAP were studied in biochemical and cell-based assays. Data presented reveal that both vGAAP and hGAAP oligomerized in a pH-dependent manner. However, the mechanisms of oligomerization are different for vGAAP and hGAAP. Disulfide bonds, and specifically cysteine residues 9 and 60, are required for vGAAP but not for hGAAP oligomerization. In functional assays, a mutant vGAAP in which oligomerization was prevented retained both its anti-apoptotic activity and its ability to modulate the Ca2+ content of intracellular stores. We conclude that monomeric GAAP can regulate both Ca2+ signaling and apoptosis.

      DISCUSSION

      Using biochemical and cell-based approaches, vGAAP and hGAAP were shown to form oligomers in vitro (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6) and in cell membranes (Fig. 8). Tetrameric GAAP was the largest oligomeric form to be readily detected (FIGURE 1, FIGURE 2), but dimers were most abundant, and monomers were also evident (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6). Further analyses are required to establish whether the relative abundance of dimers and tetramers detected in gels faithfully reflects their prevalence in cells or results from incomplete preservation of oligomers during analysis in vitro. It is, however, clear that GAAPs form homo-oligomers in cells.
      Most TMBIM proteins show several bands in SDS-PAGE suggesting that oligomerization may be a conserved property (
      • Rojas-Rivera D.
      • Armisén R.
      • Colombo A.
      • Martínez G.
      • Eguiguren A.L.
      • Díaz A.
      • Kiviluoto S.
      • Rodríguez D.
      • Patron M.
      • Rizzuto R.
      • Bultynck G.
      • Concha M.L.
      • Sierralta J.
      • Stutzin A.
      • Hetz C.
      TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.
      ,
      • Kim H.R.
      • Lee G.H.
      • Ha K.C.
      • Ahn T.
      • Moon J.Y.
      • Lee B.J.
      • Cho S.G.
      • Kim S.
      • Seo Y.R.
      • Shin Y.J.
      • Chae S.W.
      • Reed J.C.
      • Chae H.J.
      Bax Inhibitor-1 Is a pH-dependent regulator of Ca2+ channel activity in the endoplasmic reticulum.
      ). However, the determinants of oligomerization differ for vGAAP and hGAAP. The N-terminal region of vGAAP formed oligomers, whereas the equivalent region of hGAAP did not (Fig. 4B). Furthermore, oligomerization of vGAAP, but not of hGAAP, required disulfide bonds (Fig. 6, B and C). Cysteines 9 and 60 of vGAAP were essential for oligomerization of vGAAP whether assessed in vitro (Fig. 6) or in cells (Fig. 8). Despite the high level of overall sequence identity between members of the TMBIM sub-family, these cysteine residues are poorly conserved between GAAPs (supplemental Fig. S1). The mechanism by which hGAAP forms oligomers is presently unclear but clearly independent of disulfide bridges (Figs. 3B and 4B). Although it is not possible to rule out a contribution from coiled-coil, hydrophobic, ionic, or other protein-protein interaction elements in GAAP oligomerization, the results obtained by FRET show that disruption of disulfide bridges is sufficient to prevent GAAP oligomerization in the Golgi membrane.
      BI-1, another member of the TMBIM family with functional similarity to GAAP, also forms dimers and tetramers (
      • Ahn T.
      • Yun C.H.
      • Kim H.R.
      • Chae H.J.
      Cardiolipin, phosphatidylserine, and BH4 domain of Bcl-2 family regulate Ca2+/H+ antiporter activity of human Bax inhibitor-1.
      ). The mechanisms are unresolved (
      • Xu Q.
      • Reed J.C.
      Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast.
      ,
      • Bultynck G.
      • Kiviluoto S.
      • Henke N.
      • Ivanova H.
      • Schneider L.
      • Rybalchenko V.
      • Luyten T.
      • Nuyts K.
      • De Borggraeve W.
      • Bezprozvanny I.
      • Parys J.B.
      • De Smedt H.
      • Missiaen L.
      • Methner A.
      The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.
      ,
      • Kim H.R.
      • Lee G.H.
      • Ha K.C.
      • Ahn T.
      • Moon J.Y.
      • Lee B.J.
      • Cho S.G.
      • Kim S.
      • Seo Y.R.
      • Shin Y.J.
      • Chae S.W.
      • Reed J.C.
      • Chae H.J.
      Bax Inhibitor-1 Is a pH-dependent regulator of Ca2+ channel activity in the endoplasmic reticulum.
      ), but it has been proposed that the oligomeric forms mediate ion transport (
      • Bultynck G.
      • Kiviluoto S.
      • Henke N.
      • Ivanova H.
      • Schneider L.
      • Rybalchenko V.
      • Luyten T.
      • Nuyts K.
      • De Borggraeve W.
      • Bezprozvanny I.
      • Parys J.B.
      • De Smedt H.
      • Missiaen L.
      • Methner A.
      The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.
      ,
      • Ahn T.
      • Yun C.H.
      • Kim H.R.
      • Chae H.J.
      Cardiolipin, phosphatidylserine, and BH4 domain of Bcl-2 family regulate Ca2+/H+ antiporter activity of human Bax inhibitor-1.
      ). Although oligomerization of TMBIM family members appears to be a conserved feature, the mechanisms vary and there may also be different functional consequences for the different oligomeric states of TMBIM proteins.
      Our identification of point mutations that prevent oligomerization of vGAAP (FIGURE 6, FIGURE 7, FIGURE 8) allowed direct assessment of the activity of a monomeric GAAP. Surprisingly, monomeric vGAAP mimicked wild type vGAAP in both reducing the Ca2+ content of intracellular stores and in protecting cells from apoptosis (Fig. 9). There are precedents with other oligomeric proteins that retain their function as monomers (
      • Fafournoux P.
      • Noël J.
      • Pouysségur J.
      Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.
      ,
      • Karasawa A.
      • Mitsui K.
      • Matsushita M.
      • Kanazawa H.
      Intermolecular cross-linking of monomers in Helicobacter pylori Na+/H+ antiporter NhaA at the dimer interface inhibits antiporter activity.
      ,
      • Opi S.
      • Takeuchi H.
      • Kao S.
      • Khan M.A.
      • Miyagi E.
      • Goila-Gaur R.
      • Iwatani Y.
      • Levin J.G.
      • Strebel K.
      Monomeric APOBEC3G is catalytically active and has antiviral activity.
      ). In some cases, oligomerization allows differential activity (
      • Fatmi M.Q.
      • Chang C.E.
      The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase.
      ,
      • Woolf P.J.
      • Linderman J.J.
      Self organization of membrane proteins via dimerization.
      ) or trafficking (
      • Jordan B.A.
      • Trapaidze N.
      • Gomes I.
      • Nivarthi R.
      • Devi L.A.
      Oligomerization of opioid receptors with β2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation.
      ,
      • Balasubramanian S.
      • Teissére J.A.
      • Raju D.V.
      • Hall R.A.
      Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking.
      ). For example, some ion exchangers can assemble into oligomeric structures that modulate ion flux, but the oligomers are not absolutely required for function (
      • Kew J.N.
      • Davies C.H.
      ,
      • Gadsby D.C.
      Ion channels versus ion pumps: the principal difference, in principle.
      ). Oligomerization of GAAPs may likewise confer additional modulation or undefined functions, without being required to protect cells from apoptosis. It is notable that different TMBIM family proteins, GRINA and BI-1, are proposed to form hetero-oligomers that allow further regulation of activity (
      • Rojas-Rivera D.
      • Armisén R.
      • Colombo A.
      • Martínez G.
      • Eguiguren A.L.
      • Díaz A.
      • Kiviluoto S.
      • Rodríguez D.
      • Patron M.
      • Rizzuto R.
      • Bultynck G.
      • Concha M.L.
      • Sierralta J.
      • Stutzin A.
      • Hetz C.
      TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.
      ). For GAAPs, it is also conceivable that the different oligomeric forms may have additional, possibly regulatory, functions.
      Oligomerization of GAAPs and BI-1 is pH-dependent but with contrasting pH-sensitivities. Low pH favors oligomerization of BI-1 (
      • Kim H.R.
      • Lee G.H.
      • Ha K.C.
      • Ahn T.
      • Moon J.Y.
      • Lee B.J.
      • Cho S.G.
      • Kim S.
      • Seo Y.R.
      • Shin Y.J.
      • Chae S.W.
      • Reed J.C.
      • Chae H.J.
      Bax Inhibitor-1 Is a pH-dependent regulator of Ca2+ channel activity in the endoplasmic reticulum.
      ), whereas oligomerization of GAAP is more pronounced at high pH (Fig. 2). It is not yet clear whether this pH regulation, the only regulatory mechanism so far identified for GAAP, is exercised at the cytosolic or luminal surface of GAAP. Resting cytosolic pH is typically 7.2, but it is higher in solid tumor cells (
      • Neri D.
      • Supuran C.T.
      Interfering with pH regulation in tumours as a therapeutic strategy.
      ), and it is dynamically regulated by extracellular stimuli in many cells (
      • Capasso M.
      • DeCoursey T.E.
      • Dyer M.J.
      pH regulation and beyond: unanticipated functions for the voltage-gated proton channel, HVCN1.
      ). Golgi pH fluctuates between 6 and 6.7 (
      • Paroutis P.
      • Touret N.
      • Grinstein S.
      The pH of the secretory pathway: measurement, determinants, and regulation.
      ). Across these pH ranges, a substantial fraction of GAAP is monomeric (Fig. 2), consistent with Golgi-localized GAAP monomers regulating cellular behavior. However, physiological changes in cytosolic or Golgi pH may redistribute GAAP between its active monomeric form and oligomers that may be inactive or behave differently. It was reported that BI-1 oligomerization peaks at pH 5, very different from the physiological pH of ER of ∼7 (
      • Paroutis P.
      • Touret N.
      • Grinstein S.
      The pH of the secretory pathway: measurement, determinants, and regulation.
      ), at which BI-1 was shown to be monomeric (
      • Kim H.R.
      • Lee G.H.
      • Ha K.C.
      • Ahn T.
      • Moon J.Y.
      • Lee B.J.
      • Cho S.G.
      • Kim S.
      • Seo Y.R.
      • Shin Y.J.
      • Chae S.W.
      • Reed J.C.
      • Chae H.J.
      Bax Inhibitor-1 Is a pH-dependent regulator of Ca2+ channel activity in the endoplasmic reticulum.
      ). These discrepancies might suggest that the monomeric form of BI-1 could still be functional, as seen for GAAP.
      In summary, this work has shown that oligomerization of GAAPs, conserved members of the TMBIM protein family, is regulated by physiological changes in pH, and has established the determinants of oligomerization. Mutation of residues required for oligomerization of vGAAP produced a monomeric vGAAP, and use of this form established that monomeric GAAP is functional in both protecting cells from apoptosis and in reducing the Ca2+ content of the ER. This is the first demonstration that a monomeric TMBIM family member is functional.

      Acknowledgments

      We thank Dr. Theresa Ward (London School of Hygiene and Tropical Medicine) for the plasmid GRASP65-GFP, Prof. Rick Randall (University of St. Andrews, UK) for providing the HIV-1-based lentivirus system, Dr. Heike Laman (University of Cambridge, UK) for providing the bicistronic lentiviral expression vector and Mr. Nigel Miller for assistance with cell sorting.

      REFERENCES

        • Gubser C.
        • Bergamaschi D.
        • Hollinshead M.
        • Lu X.
        • van Kuppeveld F.J.
        • Smith G.L.
        A new inhibitor of apoptosis from vaccinia virus and eukaryotes.
        PLoS Pathog. 2007; 3: e17
        • Lee S.
        • Jo M.
        • Lee J.
        • Koh S.S.
        • Kim S.
        Identification of novel universal housekeeping genes by statistical analysis of microarray data.
        J. Biochem. Mol. Biol. 2007; 40: 226-231
        • van ‘t Veer L.J.
        • Dai H.
        • van de Vijver M.J.
        • He Y.D.
        • Hart A.A.
        • Mao M.
        • Peterse H.L.
        • van der Kooy K.
        • Marton M.J.
        • Witteveen A.T.
        • Schreiber G.J.
        • Kerkhoven R.M.
        • Roberts C.
        • Linsley P.S.
        • Bernards R.
        • Friend S.H.
        Gene expression profiling predicts clinical outcome of breast cancer.
        Nature. 2002; 415: 530-536
        • Hu L.
        • Smith T.F.
        • Goldberger G.
        LFG: a candidate apoptosis regulatory gene family.
        Apoptosis. 2009; 14: 1255-1265
        • Xu Q.
        • Reed J.C.
        Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast.
        Mol. Cell. 1998; 1: 337-346
        • Rojas-Rivera D.
        • Armisén R.
        • Colombo A.
        • Martínez G.
        • Eguiguren A.L.
        • Díaz A.
        • Kiviluoto S.
        • Rodríguez D.
        • Patron M.
        • Rizzuto R.
        • Bultynck G.
        • Concha M.L.
        • Sierralta J.
        • Stutzin A.
        • Hetz C.
        TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.
        Cell Death Differ. 2012; 19: 1013-1026
        • Zhou J.
        • Zhu T.
        • Hu C.
        • Li H.
        • Chen G.
        • Xu G.
        • Wang S.
        • Zhou J.
        • Ma D.
        Comparative genomics and function analysis on BI1 family.
        Comput Biol. Chem. 2008; 32: 159-162
        • Reimers K.
        • Choi C.Y.
        • Bucan V.
        • Vogt P.M.
        The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis.
        Curr. Mol. Med. 2008; 8: 148-156
        • Carrara G.
        • Saraiva N.
        • Gubser C.
        • Johnson B.F.
        • Smith G.L.
        Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family.
        J. Biol. Chem. 2012; 287: 15896-15905
        • Xu C.
        • Xu W.
        • Palmer A.E.
        • Reed J.C.
        BI-1 regulates endoplasmic reticulum Ca2+ homeostasis downstream of Bcl-2 family proteins.
        J. Biol. Chem. 2008; 283: 11477-11484
        • Ahn T.
        • Yun C.H.
        • Chae H.Z.
        • Kim H.R.
        • Chae H.J.
        Ca2+/H+ antiporter-like activity of human recombinant Bax inhibitor-1 reconstituted into liposomes.
        FEBS J. 2009; 276: 2285-2291
        • Bultynck G.
        • Kiviluoto S.
        • Henke N.
        • Ivanova H.
        • Schneider L.
        • Rybalchenko V.
        • Luyten T.
        • Nuyts K.
        • De Borggraeve W.
        • Bezprozvanny I.
        • Parys J.B.
        • De Smedt H.
        • Missiaen L.
        • Methner A.
        The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.
        J. Biol. Chem. 2012; 287: 2544-2557
        • Orrenius S.
        • Zhivotovsky B.
        • Nicotera P.
        Regulation of cell death: the calcium-apoptosis link.
        Nat. Rev. Mol. Cell Biol. 2003; 4: 552-565
        • de Mattia F.
        • Gubser C.
        • van Dommelen M.M.
        • Visch H.J.
        • Distelmaier F.
        • Postigo A.
        • Luyten T.
        • Parys J.B.
        • de Smedt H.
        • Smith G.L.
        • Willems P.H.
        • van Kuppeveld F.J.
        Human Golgi antiapoptotic protein modulates intracellular calcium fluxes.
        Mol. Biol. Cell. 2009; 20: 3638-3645
        • Doceul V.
        • Hollinshead M.
        • van der Linden L.
        • Smith G.L.
        Repulsion of superinfecting virions: a mechanism for rapid virus spread.
        Science. 2010; 327: 873-876
        • Drew D.
        • Lerch M.
        • Kunji E.
        • Slotboom D.J.
        • de Gier J.W.
        Optimization of membrane protein overexpression and purification using GFP fusions.
        Nat. Methods. 2006; 3: 303-313
        • van der Krogt G.N.
        • Ogink J.
        • Ponsioen B.
        • Jalink K.
        A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.
        PLoS One. 2008; 3: e1916
        • Groden D.L.
        • Guan Z.
        • Stokes B.T.
        Determination of Fura-2 dissociation constants following adjustment of the apparent Ca-EGTA association constant for temperature and ionic strength.
        Cell Calcium. 1991; 12: 279-287
        • Parkinson J.E.
        • Smith G.L.
        Vaccinia virus gene A36R encodes a M(r) 43–50 K protein on the surface of extracellular enveloped virus.
        Virology. 1994; 204: 376-390
        • Kornblatt J.A.
        • Lake D.F.
        Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene.
        Can J. Biochem. 1980; 58: 219-224
        • Kim H.R.
        • Lee G.H.
        • Ha K.C.
        • Ahn T.
        • Moon J.Y.
        • Lee B.J.
        • Cho S.G.
        • Kim S.
        • Seo Y.R.
        • Shin Y.J.
        • Chae S.W.
        • Reed J.C.
        • Chae H.J.
        Bax Inhibitor-1 Is a pH-dependent regulator of Ca2+ channel activity in the endoplasmic reticulum.
        J. Biol. Chem. 2008; 283: 15946-15955
        • Barr F.A.
        • Nakamura N.
        • Warren G.
        Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae.
        EMBO J. 1998; 17: 3258-3268
        • Ahn T.
        • Yun C.H.
        • Kim H.R.
        • Chae H.J.
        Cardiolipin, phosphatidylserine, and BH4 domain of Bcl-2 family regulate Ca2+/H+ antiporter activity of human Bax inhibitor-1.
        Cell Calcium. 2010; 47: 387-396
        • Fafournoux P.
        • Noël J.
        • Pouysségur J.
        Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.
        J. Biol. Chem. 1994; 269: 2589-2596
        • Karasawa A.
        • Mitsui K.
        • Matsushita M.
        • Kanazawa H.
        Intermolecular cross-linking of monomers in Helicobacter pylori Na+/H+ antiporter NhaA at the dimer interface inhibits antiporter activity.
        Biochem. J. 2010; 426: 99-108
        • Opi S.
        • Takeuchi H.
        • Kao S.
        • Khan M.A.
        • Miyagi E.
        • Goila-Gaur R.
        • Iwatani Y.
        • Levin J.G.
        • Strebel K.
        Monomeric APOBEC3G is catalytically active and has antiviral activity.
        J. Virol. 2006; 80: 4673-4682
        • Fatmi M.Q.
        • Chang C.E.
        The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase.
        PLoS Comput. Biol. 2010; 6: e1000994
        • Woolf P.J.
        • Linderman J.J.
        Self organization of membrane proteins via dimerization.
        Biophys. Chem. 2003; 104: 217-227
        • Jordan B.A.
        • Trapaidze N.
        • Gomes I.
        • Nivarthi R.
        • Devi L.A.
        Oligomerization of opioid receptors with β2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation.
        Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 343-348
        • Balasubramanian S.
        • Teissére J.A.
        • Raju D.V.
        • Hall R.A.
        Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking.
        J. Biol. Chem. 2004; 279: 18840-18850
        • Kew J.N.
        • Davies C.H.
        Ion Channels: from Structure to Function. 2nd ed. Oxford University Press, New York2010: 172-182
        • Gadsby D.C.
        Ion channels versus ion pumps: the principal difference, in principle.
        Nat. Rev. Mol. Cell Biol. 2009; 10: 344-352
        • Neri D.
        • Supuran C.T.
        Interfering with pH regulation in tumours as a therapeutic strategy.
        Nat. Rev. Drug Discov. 2011; 10: 767-777
        • Capasso M.
        • DeCoursey T.E.
        • Dyer M.J.
        pH regulation and beyond: unanticipated functions for the voltage-gated proton channel, HVCN1.
        Trends Cell Biol. 2011; 21: 20-28
        • Paroutis P.
        • Touret N.
        • Grinstein S.
        The pH of the secretory pathway: measurement, determinants, and regulation.
        Physiology. 2004; 19: 207-215