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Post-translational Membrane Insertion of Tail-anchored Transmembrane EF-hand Ca2+ Sensor Calneurons Requires the TRC40/Asna1 Protein Chaperone

2011; Elsevier BV; Volume: 286; Issue: 42 Linguagem: Inglês

10.1074/jbc.m111.280339

ISSN

1083-351X

Autores

Johannes Hradsky, Vijeta Raghuram, Parameshwar Pasham Reddy, Gemma Navarro, Mike Hupe, Vicent Casadó, Peter J. McCormick, Yogendra Sharma, Michael R. Kreutz, Marina Mikhaylova,

Tópico(s)

Neurobiology and Insect Physiology Research

Resumo

Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIβ activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. /In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca2+ sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions. Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIβ activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. /In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca2+ sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions. IntroductionCalcium (Ca2+) signaling in neurons is highly segregated both spatially and temporally. This is reflected by the broad range of phenomena, including activity-dependent gene transcription, synaptic plasticity, neurotransmitter release, and intracellular trafficking processes that are controlled by Ca2+ transients (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4116) Google Scholar, 2Mikhaylova M. Hradsky J. Kreutz M.R. J. Neurochem. 2011; 118: 695-713Crossref PubMed Scopus (47) Google Scholar). Many different Ca2+-binding proteins that belong to the EF-hand family of calmodulin (CaM) 4The abbreviations used are: CaMcalmodulinBRETbioluminescence resonance energy transferDLSdynamic light scatteringERendoplasmic reticulumFRAPfluorescence recovery after photobleachingNCSneuronal calcium sensorPI(4,5)P2phosphatidylinositol 4,5-bisphosphatePI-4KIIIβphosphatidylinositol 4-OH kinase IIIβPI(4)Pphosphatidylinositol 4-phosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-trisphosphatePMplasma membranePLAproximity ligation assayTAtail-anchoredTGNtrans-Golgi networkTMDtransmembrane domainEenhancedaaamino acidPLOprotein-lipid overlaySRPsignal recognition particleMBPmaltose-binding protein. -like Ca2+ sensors serve as essential regulators of these events. Based on the history of their discovery and their evolution, the members of this family can be divided in two groups, the neuronal calcium sensor and neuronal calcium-binding proteins (2Mikhaylova M. Hradsky J. Kreutz M.R. J. Neurochem. 2011; 118: 695-713Crossref PubMed Scopus (47) Google Scholar). Neuronal calcium-binding proteins consist of two subfamilies, Caldendrin/CaBP1–5 (2Mikhaylova M. Hradsky J. Kreutz M.R. J. Neurochem. 2011; 118: 695-713Crossref PubMed Scopus (47) Google Scholar, 3Burgoyne R.D. Nat. Rev. Neurosci. 2007; 8: 182-193Crossref PubMed Scopus (412) Google Scholar) and calneurons (also called CaBP7 and -8) (4Mikhaylova M. Sharma Y. Reissner C. Nagel F. Aravind P. Rajini B. Smalla K.H. Gundelfinger E.D. Kreutz M.R. Biochim. Biophys. Acta. 2006; 1763: 1229-1237Crossref PubMed Scopus (36) Google Scholar, 5McCue H.V. Haynes L.P. Burgoyne R.D. BMC Res. Notes. 2010; 3: 118Crossref PubMed Scopus (19) Google Scholar).A distinct subcellular localization at certain membranes is critical for the function of many CaM-like Ca2+ sensors, and neuronal calcium sensor proteins like Frequenin/neuronal calcium sensor-1 (NCS-1) and Hippocalcin are N-terminally myristoylated, which provides a lipid anchor for membrane attachment (6O'Callaghan D.W. Haynes L.P. Burgoyne R.D. Biochem. J. 2005; 391: 231-238Crossref PubMed Scopus (34) Google Scholar). Interestingly, membrane localization can be controlled by a Ca2+-myristoyl switch. In the case of Hippocalcin, binding of Ca2+ induces a conformational change and exposure of a hydrophobic myristoyl tail and subsequent translocation of the protein to the plasma membrane (PM) and the Golgi complex (7O'Callaghan D.W. Tepikin A.V. Burgoyne R.D. J. Cell Biol. 2003; 163: 715-721Crossref PubMed Scopus (67) Google Scholar). Association with these particular membrane compartments is controlled by a direct interaction of Hippocalcin with phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (6O'Callaghan D.W. Haynes L.P. Burgoyne R.D. Biochem. J. 2005; 391: 231-238Crossref PubMed Scopus (34) Google Scholar). Phosphoinositides contribute to the unique identity of organelles, and binding of PI(4)P, specific for the trans-Golgi network (TGN), and PI(4,5)P2 at the plasma membrane might explain at least in part the targeting of Hippocalcin and NCS-1 to these membranes (6O'Callaghan D.W. Haynes L.P. Burgoyne R.D. Biochem. J. 2005; 391: 231-238Crossref PubMed Scopus (34) Google Scholar).The Golgi by itself is a Ca2+ store that contains release and sequestration apparatus, and several studies have shown that Ca2+ regulates the passage of proteins along the secretory pathway as well as the exit of vesicles from the TGN (8Dolman N.J. Tepikin A.V. Cell Calcium. 2006; 40: 505-512Crossref PubMed Scopus (47) Google Scholar, 9Micaroni M. Curr. Mol. Med. 2010; 10: 763-773Crossref PubMed Scopus (20) Google Scholar). The local synthesis of PI(4)P and PI(4,5)P2 is crucial for TGN to PM trafficking, and the activity of phosphatidylinositol 4-OH kinase III β (PI-4KIIIβ) at the Golgi membrane is the first mandatory step in this process (10Balla A. Balla T. Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Interestingly, the enzymatic activity of PI-4KIIIβ is regulated by an interaction with NCS-1 and calneurons. At low Ca2+ levels, PI-4KIIIβ is preferentially associated with calneurons, whereas high Ca2+ levels favor binding of NCS-1, and in sharp contrast to the activating role of NCS-1, calneurons strongly inhibit PI-4KIIIβ activity with markedly attenuated PI-4KIIIβ activity at low to medium Ca2+ levels (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar). It was therefore suggested that calneurons operate as a filter that suppresses PI-4KIIIβ activity at submaximal amplitudes of Golgi Ca2+ transients and thereby provides a tonic inhibition that is only released under conditions of sustained Ca2+ release in secretory cells (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar, 12Mikhaylova M. Reddy P.P. Kreutz M.R. Biochem. Soc. Trans. 2010; 38: 177-180Crossref PubMed Scopus (5) Google Scholar).Calneurons are highly abundant at the Golgi apparatus in neurons, and their Golgi association is much more prominent than those of other calcium sensor proteins like Caldendrin and NCS-1 (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar, 13McCue H.V. Burgoyne R.D. Haynes L.P. Biochem. Biophys. Res. Commun. 2009; 380: 825-831Crossref PubMed Scopus (18) Google Scholar). Structurally, calneurons possess four EF-hand motifs out of which EF-hands three and four are nonfunctional in the sense that they do not bind Ca2+ (Fig. 1A). Although they are efficiently localized to the TGN, calneurons do not contain an N-myristoylation motif that could provide them with a lipid membrane anchor. Thus, the mechanism by which calneurons can be localized to the TGN is unclear. Recently, it was suggested that they are transmembrane proteins and that the membrane localization of calneurons might be provided by the C-terminal hydrophobic region that serves as the transmembrane domain (TMD), with the N terminus oriented toward the cytosol (Fig. 1A) (14McCue H.V. Burgoyne R.D. Haynes L.P. PLoS One. 2011; 6: e17853Crossref PubMed Scopus (12) Google Scholar). In this study, we aimed to identify the mechanisms that target calneurons to the TGN.EXPERIMENTAL PROCEDURESInformation about the plasmids and antibodies used in this study as well as a detailed description of subcellular fractionation of HeLa cells and quantification for the Golgi localization of calneuron-1 constructs can be found in the supplemental material.Cell Culture, Transient Transfection, and ImmunostainingHEK-293T, COS-7, and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mm l-glutamine, 100 units/ml penicillin/streptomycin, and 5% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen). For immunofluorescence studies and live imaging experiments, COS-7 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. 24 h after transfection, the cells were fixed with 4% paraformaldehyde and processed as described previously (15Dieterich D.C. Karpova A. Mikhaylova M. Zdobnova I. König I. Landwehr M. Kreutz M. Smalla K.H. Richter K. Landgraf P. Reissner C. Boeckers T.M. Zuschratter W. Spilker C. Seidenbecher C.I. Garner C.C. Gundelfinger E.D. Kreutz M.R. PLoS Biol. 2008; 6: e34Crossref PubMed Scopus (134) Google Scholar). Except for the GFP, live staining COS-7 cells were preincubated with anti-GFP rabbit antibody (1:200) diluted in DMEM growth media for 1 h at 37 °C and then fixed, washed with PBS, and processed for immunostaining. For the bimolecular fluorescence complementation assay, COS-7 cells were co-transfected with equal amounts of the fusion constructs containing the N- and C-terminal halves of split YFP variant Venus. 40 h after transfection, cells were fixed and processed for immunostaining. HEK-293T cells for BRET experiments were transfected with the corresponding fusion protein cDNAs by polyethyleneimine (Sigma) as described previously (16Carriba P. Navarro G. Ciruela F. Ferré S. Casadó V. Agnati L. Cortés A. Mallol J. Fuxe K. Canela E.I. Lluís C. Franco R. Nat. Methods. 2008; 5: 727-733Crossref PubMed Scopus (248) Google Scholar).Protein Expression, Purification, and ImmunoblottingHis6-SUMO-, GST-, and MBP-tagged fusion proteins were purified from isopropyl 1-thio-β-d-galactopyranoside-induced Escherichia coli BL21(DE3) as reported previously (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar, 17Vilardi F. Lorenz H. Dobberstein B. J. Cell Sci. 2011; 124: 1301-1307Crossref PubMed Scopus (82) Google Scholar). Alternatively to the ProBondTM purification system (Invitrogen), His6-SUMO constructs were also purified on 1-ml HisTrapHP columns (GE Healthcare) according to the manufacturer's manual. Furthermore, gel filtrations on HiLoad SuperdexTM 75 16/60 or SuperdexTM 75 10/300 GL columns (GE Healthcare) were performed for these constructs. Purified proteins were finally concentrated using Amicon 15 centrifugal filter devices (Millipore, Schwalbach, Germany) of 3- or 10-kDa cutoffs. SDS-PAGE, semi-native SDS-PAGE (containing 0.5% SDS), and native PAGE were followed by immunoblotting that was performed as described previously (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar). For dimerization studies, 1× Native Sample Buffer was used; Semi-Native Sample Buffer was prepared from 1× Native Sample Buffer and additionally contained 0.5% SDS.Liposome Sedimentation AssaysLiposome sedimentation assays were done using liposomes obtained from bovine brain fraction (Folch fraction 1, Sigma) and purified His6SUMO-calneuron-1 proteins as described previously (18Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. Science. 2004; 303: 495-499Crossref PubMed Scopus (1334) Google Scholar). All steps were carried out in the presence of either 100 μm CaCl2 or 2 mm EDTA.Size Exclusion ChromatographyEqual amounts of protein samples incubated with either 500 μm Ca2+ or 500 μm EDTA were loaded on a SuperdexTM 200 HR 10/30 column (GE Healthcare) and pre-equilibrated with a buffer containing 50 mm Tris, pH 7.5, 100 mm KCl, and either 1 mm Ca2+ or 1 mm EDTA, respectively. The separation was performed at room temperature using a flow rate of 0.5 ml/min and monitored at 280 nm. Gel filtration standards from Bio-Rad were used for both calibration and determination of void volume.Dynamic Light Scattering (DLS)DLS experiments were performed in the presence of either 500 μm Ca2+ or 500 μm EDTA on a Flexible Correlator Photocor-FC (Photocor Instruments. Inc.) with a 632.8 nm laser at 25 °C. The software, Alango DynaLS version 2.0, provided with the instrument was used to analyze the data thus obtained and calculate the mean hydrodynamic radius using the Stokes-Einstein equation, Dt = kBT/6πηRH.Co-immunoprecipitation (Co-IP) and Pulldown AssayHeterologous co-IP was performed with extracts from double transfected COS-7 cells using the μMACSTM GFP isolation kit (Miltenyi Biotec GmbH, Germany) according to the manufacturer's protocol. Eluted samples were checked on SDS-PAGE/Western blot using anti-calneuron-1 rabbit (1:300, ProteinTech) antibody. For pulldown assay experiments, COS-7 cells were transfected with different EGFP-calneuron-1 constructs. GST-TRC40/Asna1 or GST-coupled Sepharose beads were incubated with COS-7 cell extracts overnight at 4 °C. Experiments were carried out as described previously (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar). The pulldown assay with MBP-calneuron-1 coupled beads was performed in the presence of either 2 mm EDTA or 100 μm Ca2+. Detection on the immunoblot was done using anti-GFP mouse antibody (1:5000, Covance).Protein-Lipid Overlay (PLO) AssayFor the PLO assay, COS-7 cells were transfected with untagged calneuron-1, and a cell extract (extraction buffer: 1× TBS buffer containing 0.25% of Tween 20 and a protease inhibitor mixture, Roche Applied Science) was prepared 48 h after transfection. The lipid strips containing different lipids (Lipid Strips and PIP Strips from Echelon Biosciences (Mobitec, Goettingen, Germany) were incubated with a COS-7 cell extract at 4 °C overnight and then developed with an anti-calneuron-1 rabbit antibody.Protein-Protein Overlay AssayDifferent recombinant His6-SUMO-calneuron-1 proteins or His6-SUMO control were diluted in 1× Native Sample Buffer also containing 0.5% SDS and 0.2% β-mercaptoethanol, subjected to SDS-PAGE (5 μg/lane), transferred onto the nitrocellulose membrane, blocked in 5% nonfat milk in TBS-T, and incubated at 4 °C overnight with recombinant GST-TRC40/Asna1 (10 μg/ml). After extensive washing, the membrane was incubated with anti-Asna1 mouse antibody following standard protocols.BRET AssaysBRET assays were performed in HEK-293T cells transiently co-transfected with a constant amount of cDNA encoding for the protein fused to Rluc and increasing amounts of cDNA corresponding to the protein fused to YFP exactly as described previously (19Navarro G. Aymerich M.S. Marcellino D. Cortés A. Casadó V. Mallol J. Canela E.I. Agnati L. Woods A.S. Fuxe K. Lluís C. Lanciego J.L. Ferré S. Franco R. J. Biol. Chem. 2009; 284: 28058-28068Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Specifications of particular experiments can be found in the supplement material.Laser Scanning Microscopy, FRAP Experiments, and Image AnalysisAll fluorescence images were obtained on a TCS SP5 II confocal laser scanning microscope (Leica, Germany) using a 63× oil objective and zoom factors in the range of 1–4×. For FRAP experiments, COS-7 cells were co-transfected with pEGFP-C1-calneuron-1 and pDsRed-Monomeric-Golgi or pGFPC1-Sec61β. 24 h later DMEM was replaced by KD buffer, and cells were placed under a confocal laser scanning microscope. A 568 and 488 nm laser line was used to monitor the DsRed channel and a 488 nm laser line for both imaging of EGFP and photobleaching. A 63× oil objective and 4× confocal zoom were used. Single plane images were recorded every 20 s for the ER marker and every minute for the calneuron-1 at the Golgi complex. Photobleaching was performed using the FRAP Wizard mode with 488 nm laser at maximum efficiency. Regions of interest of comparable size were taken for each experiment. For the evaluation of % of FRAP, the initial fluorescence was taken as 100%, and data were graphically plotted. Images were analyzed using ImageJ software (National Institutes of Health).Brefeldin A AssayCOS-7 cells were co-transfected with untagged calneuron-1, GFP-Sec61β, and DsRed-Monomeric-Golgi for 24 h. Inhibition of ER-to-Golgi trafficking was induced by prolonged (3 h) incubation with brefeldin A (Cell Signaling, New England Biolabs) at a concentration of 100 ng/ml. Control cells were treated with DMSO. Thereafter, cells were fixed and stained with an anti-calneuron-1 rabbit antibody.VSV-G Trafficking AssayCOS-7 cells were grown on coverslips and co-transfected with a VSV-G-GFP (ts045) expression plasmid and pcDNA3.1 encoding untagged calneuron-1. The transfected cells were transferred to 39.5 °C 4 h post-transfection and incubated overnight. Accumulation of VSV-G-GFP at the Golgi was induced by shifting the temperature to 20 °C for 2 h. Then the Golgi block was removed, and Golgi-to-PM trafficking was allowed by incubating the coverslips at 32 °C for 20 min, followed by formaldehyde fixation and immunostaining.Proximity Ligation Assay (PLA)PLA was performed using the Duolink II system (Olink Biosciences, Sweden) with anti-rabbit minus and anti-mouse plus probes according to the manufacturer's protocol. COS-7 cells were co-transfected with EYFP-TRC40/ASNA-1 and untagged full-length calneuron-1 or with EGFP control plasmid and calneuron-1. 24 h after transfection, cells were fixed and incubated with primary anti-ASNA mouse (1:1500) and anti-calneuron-1 rabbit antibody (1:1000, ProteinTech) or anti-GFP mouse (1:200, BAPCO) and anti-calneuron-1 rabbit antibody as control. Thereafter, the pair of oligonucleotide-labeled secondary antibodies (PLA probes) was applied on the same samples.Statistical AnalysisStatistical analysis was performed with the SPSS Statistics software (IBM, Ehningen, Germany). One-way analysis of variance was used to compare individual groups.DISCUSSIONIn previous work, we have demonstrated that both endogenous and overexpressed calneuron-1 and -2 are localized at the TGN in pyramidal neurons as well as in secretory cells (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar). The localization of calneurons tightly correlates with their function as "calcium threshold filters" for activation of PI-4KIIIβ, production of PI(4)P, and regulation of vesicular trafficking from the TGN to the plasma membrane. Shih et al. (20Shih P.Y. Lin C.L. Cheng P.W. Liao J.H. Pan C.Y. Biochem. Biophys. Res. Commun. 2009; 388: 549-553Crossref PubMed Scopus (12) Google Scholar) have recently shown that a fraction of calneuron-1 resides at the PM where calneuron-1 is involved in inhibition of N-type Ca2+ channels. Interestingly, the presence of the hydrophobic C-terminal fragment is required to induce this inhibition (20Shih P.Y. Lin C.L. Cheng P.W. Liao J.H. Pan C.Y. Biochem. Biophys. Res. Commun. 2009; 388: 549-553Crossref PubMed Scopus (12) Google Scholar). In this study, we addressed two questions important for the understanding of calneuron biosynthesis and function at the TGN and PM. Why are these proteins enriched at the TGN and which route of post-translation insertion do they utilize?It was previously suggested that calneurons are transmembrane proteins, which is an uncommon feature for CaM-like EF-hand Ca2+ sensors (14McCue H.V. Burgoyne R.D. Haynes L.P. PLoS One. 2011; 6: e17853Crossref PubMed Scopus (12) Google Scholar). We could confirm these findings and found further evidence that they are indeed TA proteins. However, in accordance with the requirement for TRC40/Asna1 binding for membrane insertion and TGN membrane thickness, the TMD region and minimal TGN targeting sequence appears to be longer than those published previously (13McCue H.V. Burgoyne R.D. Haynes L.P. Biochem. Biophys. Res. Commun. 2009; 380: 825-831Crossref PubMed Scopus (18) Google Scholar, 14McCue H.V. Burgoyne R.D. Haynes L.P. PLoS One. 2011; 6: e17853Crossref PubMed Scopus (12) Google Scholar). The joint feature of the heterogeneous group of TA proteins is that they harbor a TMD at their C terminus and undergo post-translation insertion into different membrane organelles, including the outer membrane of bacteria, membranes of mitochondria, and chloroplasts, peroxisomes, and ER membranes (41Renthal R. Cell. Mol. Life Sci. 2010; 67: 1077-1088Crossref PubMed Scopus (31) Google Scholar). Once TA proteins are integrated into the ER membrane, they can also be sorted to other membranes within the secretory pathway (42Borgese N. Brambillasca S. Colombo S. Curr. Opin. Cell Biol. 2007; 19: 368-375Crossref PubMed Scopus (153) Google Scholar). TA proteins are always oriented in the membrane with the larger N-terminal region facing the cytosol. This region is usually important for the biological function of the protein (23Rabu C. Schmid V. Schwappach B. High S. J. Cell Sci. 2009; 122: 3605-3612Crossref PubMed Scopus (92) Google Scholar). In the case of calneurons, the EF-hand domains are exposed to the cytosol where they are involved in Ca2+-dependent regulation of vesicular trafficking at the TGN (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar, 14McCue H.V. Burgoyne R.D. Haynes L.P. PLoS One. 2011; 6: e17853Crossref PubMed Scopus (12) Google Scholar) and inhibition of N-type Ca2+ channels (20Shih P.Y. Lin C.L. Cheng P.W. Liao J.H. Pan C.Y. Biochem. Biophys. Res. Commun. 2009; 388: 549-553Crossref PubMed Scopus (12) Google Scholar) at the PM. Trafficking from the TGN to PM appears to follow the route of VSV-G. In turn, this provides a unique mechanism for a highly restricted localization of these Ca2+ sensor proteins, and calneuron-2 indeed appears to be exclusively situated at HeLa cell membranes.In addition, we found that the 23-aa-long TMD of calneuron-1 is necessary and sufficient for TGN localization of the protein. The previously suggested 17-aa shorter TMD region (13McCue H.V. Burgoyne R.D. Haynes L.P. Biochem. Biophys. Res. Commun. 2009; 380: 825-831Crossref PubMed Scopus (18) Google Scholar) co-localized less efficiently with the Golgi marker GM130, and we observed a more diffuse distribution that is commonly observed for ER proteins or proteins present in the cytosol. It has been shown previously that information decoded in the length and the hydrophobicity of the TMD defines the destination of TA proteins (20Shih P.Y. Lin C.L. Cheng P.W. Liao J.H. Pan C.Y. Biochem. Biophys. Res. Commun. 2009; 388: 549-553Crossref PubMed Scopus (12) Google Scholar, 32Favaloro V. Vilardi F. Schlecht R. Mayer M.P. Dobberstein B. J. Cell Sci. 2010; 123: 1522-1530Crossref PubMed Scopus (47) Google Scholar, 42Borgese N. Brambillasca S. Colombo S. Curr. Opin. Cell Biol. 2007; 19: 368-375Crossref PubMed Scopus (153) Google Scholar, 43Horie C. Suzuki H. Sakaguchi M. Mihara K. Mol. Biol. Cell. 2002; 13: 1615-1625Crossref PubMed Scopus (122) Google Scholar). The reason why calneuron-1_CT and calneuron-1_23aa are targeted to the TGN more efficiently than the shorter TMD probably relates to the properties of the TGN membrane bilayers. Membranes of different intracellular compartments have different lipid compositions that result in increasing thickness and decreasing fluidity of the bilayers from the ER-to-Golgi complex and the PM (44Ceppi P. Colombo S. Francolini M. Raimondo F. Borgese N. Masserini M. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 16269-16274Crossref PubMed Scopus (25) Google Scholar). Glycerophospholipids and sphingolipids are initially synthesized in the ER and then transferred between different compartments (45Klemm R.W. Ejsing C.S. Surma M.A. Kaiser H.J. Gerl M.J. Sampaio J.L. de Robillard Q. Ferguson C. Proszynski T.J. Shevchenko A. Simons K. J. Cell Biol. 2009; 185: 601-612Crossref PubMed Scopus (298) Google Scholar, 46Patterson G.H. Hirschberg K. Polishchuk R.S. Gerlich D. Phair R.D. Lippincott-Schwartz J. Cell. 2008; 133: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Lippincott-Schwartz and co-workers (46Patterson G.H. Hirschberg K. Polishchuk R.S. Gerlich D. Phair R.D. Lippincott-Schwartz J. Cell. 2008; 133: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) suggested the so-called "rapid partitioning model." Based upon the cellular glycerophospholipids and sphingolipid compositions of ER, Golgi, and plasma membranes, they propose a steady state cis-to-trans gradient in sphingolipid/glycerophospholipid ratio across the Golgi stack (46Patterson G.H. Hirschberg K. Polishchuk R.S. Gerlich D. Phair R.D. Lippincott-Schwartz J. Cell. 2008; 133: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Several lines of evidence suggest that TGN membranes are thicker than ER membranes (46Patterson G.H. Hirschberg K. Polishchuk R.S. Gerlich D. Phair R.D. Lippincott-Schwartz J. Cell. 2008; 133: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 47Holthuis J.C. Pomorski T. Raggers R.J. Sprong H. Van Meer G. Physiol. Rev. 2001; 81: 1689-1723Crossref PubMed Scopus (252) Google Scholar), and a number of proteins depend on features of their TMD for correct sorting between the ER and the Golgi apparatus (46Patterson G.H. Hirschberg K. Polishchuk R.S. Gerlich D. Phair R.D. Lippincott-Schwartz J. Cell. 2008; 133: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 48Lundbaek J.A. Andersen O.S. Werge T. Nielsen C. Biophys. J. 2003; 84: 2080-2089Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). TA proteins frequently require short and moderately hydrophobic TMDs to remain resident at the ER, where they are excluded from the more ordered bilayer regions that get transported to the Golgi (49Borgese N. Colombo S. Pedrazzini E. J. Cell Biol. 2003; 161: 1013-1019Crossref PubMed Scopus (203) Google Scholar). For example, Ceppi et al. (44Ceppi P. Colombo S. Francolini M. Raimondo F. Borgese N. Masserini M. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 16269-16274Crossref PubMed Scopus (25) Google Scholar) could demonstrate that the wild type form of the ER resident TA protein cytochrome b5 with a 17-aa TMD could be found at the PM when five extra nonpolar amino acids were fused to extend the transmembranal helix. Apart from the longer TMD, another reason for the localization of calneurons to the post-ER components of the secretory trafficking pathway is probably the interaction of calneuron-1 with phospholipids enriched in TGN, endosomes, and the PM. We found specific binding to PI(4)P-phosphoinositide, a lipid characteristic for the TGN (50Behnia R. Munro S. Nature. 2005; 438: 597-604Crossref PubMed Scopus (370) Google Scholar). PI(4)P is a lipid product of TGN localized PI-4KIIIβ (10Balla A. Balla T. Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) that is required to recruit several effectors to promote the budding and fission of Golgi-derived transport vesicles (10Balla A. Balla T. Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Moreover, PI(4)P is a precursor of PI(4,5)P2, which in turn is enriched at the PM; and in a PLO assay, we also found binding of calneuron-1 to this phosphoinositide.TA proteins are first inserted into the ER membranes and, later on, depending upon their features, distributed to other compartments of the secretory pathway. With blocking of ER-to-Golgi protein transport by applying brefeldin A and with independent FRAP experiments, we could show that EGFP-calneuron-1 is indeed transported from the ER to the Golgi. Moreover, we show that calneuron-1 strongly co-localizes with VSV-G post-Golgi clusters 20 min after removing the Golgi block, suggesting that fraction of the protein follows the route of VSV-G trafficking from ER-to-plasma membranes. The highly hydrophobic TA core of calneurons as expected requires assistance of chaperones for the integration into the ER membrane (23Rabu C. Schmid V. Schwappach B. High S. J. Cell Sci. 2009; 122: 3605-3612Crossref PubMed Scopus (92) Google Scholar). Calneuron-1 interacts with the TRC40/Asna1 complex in vitro and in vivo, and this complex only binds the monomeric form of calneuron-1. Of note, we also observed a weak binding between calneuron-1 and Sec61β, one of the subunits of Sec61 protein translocation channel associated with SRP complex. Interestingly, Sec61β is actually a TA protein by itself and it is inserted into the ER membranes via TRC40/Asna1 (30Stefanovic S. Hegde R.S. Cell. 2007; 128: 1147-1159Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). SRP-dependent delivery of TA proteins to the ER has been reported previously, and this route has been shown to act as a complementary pathway for TA proteins such as Syb2 and Sec61β (51Abell B.M. Pool M.R. Schlenker O. Sinning I. High S. EMBO J. 2004; 23: 2755-2764Crossref PubMed Scopus (106) Google Scholar). Therefore, the possibility that calneuron-1 in the absence of TRC40/Asna1 might also utilize the SRP-dependent insertion mechanism cannot be excluded.Finally, we found that calneurons dimerize and potentially multimerize in vitro and in vivo. We found no evidence that a parallel dimer might give rise to a cytosolic pool that is not accessible for TRC40/Asna1. Instead, the dimer appears to be present at the TGN and potentially in later steps of the secretory pathway. Interestingly, we found that the N-terminal domain lacking the TMD is capable of dimerization by itself. At present, it is therefore unclear how the membranous dimer is formed, but it has been shown that dimerization of the CaM-like part can play an important role for the function of EF-hand Ca2+ sensors (36Lusin J.D. Vanarotti M. Li C. Valiveti A. Ames J.B. Biochemistry. 2008; 47: 2252-2264Crossref PubMed Scopus (41) Google Scholar). A calneuron dimer or multimer might also regulate the retention of the protein at the TGN membrane as has been shown for other membrane proteins (52Sasai K. Ikeda Y. Tsuda T. Ihara H. Korekane H. Shiota K. Taniguchi N. J. Biol. Chem. 2001; 276: 759-765Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 53Lopez-Gimenez J.F. Canals M. Pediani J.D. Milligan G. Mol. Pharmacol. 2007; 71: 1015-1029Crossref PubMed Scopus (150) Google Scholar). It is also conceivable that self-association of calneurons can influence membrane curvature, which has been shown for many other proteins with a large cytosolic domain and a membrane spanning region (18Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. Science. 2004; 303: 495-499Crossref PubMed Scopus (1334) Google Scholar, 54McMahon H.T. Gallop J.L. Nature. 2005; 438: 590-596Crossref PubMed Scopus (1575) Google Scholar, 55Stachowiak J.C. Hayden C.C. Sasaki D.Y. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 7781-7786Crossref PubMed Scopus (152) Google Scholar). IntroductionCalcium (Ca2+) signaling in neurons is highly segregated both spatially and temporally. This is reflected by the broad range of phenomena, including activity-dependent gene transcription, synaptic plasticity, neurotransmitter release, and intracellular trafficking processes that are controlled by Ca2+ transients (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4116) Google Scholar, 2Mikhaylova M. Hradsky J. Kreutz M.R. J. Neurochem. 2011; 118: 695-713Crossref PubMed Scopus (47) Google Scholar). Many different Ca2+-binding proteins that belong to the EF-hand family of calmodulin (CaM) 4The abbreviations used are: CaMcalmodulinBRETbioluminescence resonance energy transferDLSdynamic light scatteringERendoplasmic reticulumFRAPfluorescence recovery after photobleachingNCSneuronal calcium sensorPI(4,5)P2phosphatidylinositol 4,5-bisphosphatePI-4KIIIβphosphatidylinositol 4-OH kinase IIIβPI(4)Pphosphatidylinositol 4-phosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-trisphosphatePMplasma membranePLAproximity ligation assayTAtail-anchoredTGNtrans-Golgi networkTMDtransmembrane domainEenhancedaaamino acidPLOprotein-lipid overlaySRPsignal recognition particleMBPmaltose-binding protein. -like Ca2+ sensors serve as essential regulators of these events. Based on the history of their discovery and their evolution, the members of this family can be divided in two groups, the neuronal calcium sensor and neuronal calcium-binding proteins (2Mikhaylova M. Hradsky J. Kreutz M.R. J. Neurochem. 2011; 118: 695-713Crossref PubMed Scopus (47) Google Scholar). Neuronal calcium-binding proteins consist of two subfamilies, Caldendrin/CaBP1–5 (2Mikhaylova M. Hradsky J. Kreutz M.R. J. Neurochem. 2011; 118: 695-713Crossref PubMed Scopus (47) Google Scholar, 3Burgoyne R.D. Nat. Rev. Neurosci. 2007; 8: 182-193Crossref PubMed Scopus (412) Google Scholar) and calneurons (also called CaBP7 and -8) (4Mikhaylova M. Sharma Y. Reissner C. Nagel F. Aravind P. Rajini B. Smalla K.H. Gundelfinger E.D. Kreutz M.R. Biochim. Biophys. Acta. 2006; 1763: 1229-1237Crossref PubMed Scopus (36) Google Scholar, 5McCue H.V. Haynes L.P. Burgoyne R.D. BMC Res. Notes. 2010; 3: 118Crossref PubMed Scopus (19) Google Scholar).A distinct subcellular localization at certain membranes is critical for the function of many CaM-like Ca2+ sensors, and neuronal calcium sensor proteins like Frequenin/neuronal calcium sensor-1 (NCS-1) and Hippocalcin are N-terminally myristoylated, which provides a lipid anchor for membrane attachment (6O'Callaghan D.W. Haynes L.P. Burgoyne R.D. Biochem. J. 2005; 391: 231-238Crossref PubMed Scopus (34) Google Scholar). Interestingly, membrane localization can be controlled by a Ca2+-myristoyl switch. In the case of Hippocalcin, binding of Ca2+ induces a conformational change and exposure of a hydrophobic myristoyl tail and subsequent translocation of the protein to the plasma membrane (PM) and the Golgi complex (7O'Callaghan D.W. Tepikin A.V. Burgoyne R.D. J. Cell Biol. 2003; 163: 715-721Crossref PubMed Scopus (67) Google Scholar). Association with these particular membrane compartments is controlled by a direct interaction of Hippocalcin with phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (6O'Callaghan D.W. Haynes L.P. Burgoyne R.D. Biochem. J. 2005; 391: 231-238Crossref PubMed Scopus (34) Google Scholar). Phosphoinositides contribute to the unique identity of organelles, and binding of PI(4)P, specific for the trans-Golgi network (TGN), and PI(4,5)P2 at the plasma membrane might explain at least in part the targeting of Hippocalcin and NCS-1 to these membranes (6O'Callaghan D.W. Haynes L.P. Burgoyne R.D. Biochem. J. 2005; 391: 231-238Crossref PubMed Scopus (34) Google Scholar).The Golgi by itself is a Ca2+ store that contains release and sequestration apparatus, and several studies have shown that Ca2+ regulates the passage of proteins along the secretory pathway as well as the exit of vesicles from the TGN (8Dolman N.J. Tepikin A.V. Cell Calcium. 2006; 40: 505-512Crossref PubMed Scopus (47) Google Scholar, 9Micaroni M. Curr. Mol. Med. 2010; 10: 763-773Crossref PubMed Scopus (20) Google Scholar). The local synthesis of PI(4)P and PI(4,5)P2 is crucial for TGN to PM trafficking, and the activity of phosphatidylinositol 4-OH kinase III β (PI-4KIIIβ) at the Golgi membrane is the first mandatory step in this process (10Balla A. Balla T. Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Interestingly, the enzymatic activity of PI-4KIIIβ is regulated by an interaction with NCS-1 and calneurons. At low Ca2+ levels, PI-4KIIIβ is preferentially associated with calneurons, whereas high Ca2+ levels favor binding of NCS-1, and in sharp contrast to the activating role of NCS-1, calneurons strongly inhibit PI-4KIIIβ activity with markedly attenuated PI-4KIIIβ activity at low to medium Ca2+ levels (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar). It was therefore suggested that calneurons operate as a filter that suppresses PI-4KIIIβ activity at submaximal amplitudes of Golgi Ca2+ transients and thereby provides a tonic inhibition that is only released under conditions of sustained Ca2+ release in secretory cells (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar, 12Mikhaylova M. Reddy P.P. Kreutz M.R. Biochem. Soc. Trans. 2010; 38: 177-180Crossref PubMed Scopus (5) Google Scholar).Calneurons are highly abundant at the Golgi apparatus in neurons, and their Golgi association is much more prominent than those of other calcium sensor proteins like Caldendrin and NCS-1 (11Mikhaylova M. Reddy P.P. Munsch T. Landgraf P. Suman S.K. Smalla K.H. Gundelfinger E.D. Sharma Y. Kreutz M.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9093-9098Crossref PubMed Scopus (51) Google Scholar, 13McCue H.V. Burgoyne R.D. Haynes L.P. Biochem. Biophys. Res. Commun. 2009; 380: 825-831Crossref PubMed Scopus (18) Google Scholar). Structurally, calneurons possess four EF-hand motifs out of which EF-hands three and four are nonfunctional in the sense that they do not bind Ca2+ (Fig. 1A). Although they are efficiently localized to the TGN, calneurons do not contain an N-myristoylation motif that could provide them with a lipid membrane anchor. Thus, the mechanism by which calneurons can be localized to the TGN is unclear. Recently, it was suggested that they are transmembrane proteins and that the membrane localization of calneurons might be provided by the C-terminal hydrophobic region that serves as the transmembrane domain (TMD), with the N terminus oriented toward the cytosol (Fig. 1A) (14McCue H.V. Burgoyne R.D. Haynes L.P. PLoS One. 2011; 6: e17853Crossref PubMed Scopus (12) Google Scholar). In this study, we aimed to identify the mechanisms that target calneurons to the TGN.

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