The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.
References
[1]
Rambourg A, Clermont Y, Hermo L, Segretain D (1987) Tridimensional structure of the Golgi apparatus of nonciliated epithelial cells of the ductuli efferentes in rat: an electron microscope stereoscopic study. Biol Cell 60: 103–115. doi:10.1111/j.1768-322X.1987.tb00550.x. PubMed: 2825891.
[2]
Rios RM, Bornens M (2003) The Golgi apparatus at the cell centre. Curr Opin Cell Biol 15: 60–66. doi:10.1016/S0955-0674(02)00013-3. PubMed: 12517705.
[3]
Lucocq JM, Warren G (2007) Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells. EMBO J 6: 3239–3246. PubMed: 3428259.
[4]
Carcedo CH (2004) Mitotic Golgi Partitioning Is Driven by the Membrane-Fissioning Protein CtBP3/BARS. Science 305: 93–96. doi:10.1126/science.1097775. PubMed: 15232108.
[5]
Sütterlin C, Hsu P, Mallabiabarrena A, Malhotra V (2002) Fragmentation and dispersal of the pericentriolar Golgi complex is required for entry into mitosis in mammalian cells. Cell 109: 359–369. doi:10.1016/S0092-8674(02)00720-1. PubMed: 12015985.
[6]
Kodani A, Sütterlin C (2009) A new function for an old organelle: microtubule nucleation at the Golgi apparatus. EMBO J 28: 995–996. doi:10.1038/emboj.2009.85. PubMed: 19384347.
[7]
Kodani A, Sütterlin C (2008) The Golgi protein GM130 regulates centrosome morphology and function. Mol Biol Cell 19: 745–753. doi:10.1091/mbc.E07-08-0847. PubMed: 18045989.
[8]
Miserey-Lenkei S, Cou？del-Courteille A, Del Nery E, Bardin S, Piel M et al. (2006) A role for the Rab6A' GTPase in the inactivation of the Mad2-spindle checkpoint. EMBO J 25: 278–289. doi:10.1038/sj.emboj.7600929. PubMed: 16395330.
[9]
Kittler R, Pelletier L, Heninger A-K, Slabicki M, Theis M et al. (2007) Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat Cell Biol 9: 1401–1412. doi:10.1038/ncb1659. PubMed: 17994010.
[10]
Hutchins JRA, Toyoda Y, Hegemann B, Poser I, Hériché J-K et al. (2010) Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science 328: 593–599. doi:10.1126/science.1181348. PubMed: 20360068.
[11]
Tusnády GE, Simon I (1998) Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283: 489–506. doi:10.1006/jmbi.1998.2107. PubMed: 9769220.
[12]
Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17: 849–850. doi:10.1093/bioinformatics/17.9.849. PubMed: 11590105.
[13]
Larkin MA, Blackshields G, Blackshields G, Brown NP et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948. doi:10.1093/bioinformatics/btm404. PubMed: 17846036.
[14]
Goujon M, McWilliam H, Li W, Valentin F, Squizzato S et al. (2010) A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38: W695–W699. doi:10.1093/nar/gkq313. PubMed: 20439314.
[15]
Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B et al. (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47: 6172–6176. doi:10.1002/anie.200802376. PubMed: 18646237.
[16]
van de Linde S, L？schberger A, Klein T, Heidbreder M, Wolter S et al. (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6: 991–1009. doi:10.1038/nprot.2011.336. PubMed: 21720313.
[17]
Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8: 1027–1036. doi:10.1038/nmeth.1768. PubMed: 22056676.
[18]
Lorenz H, Hailey DW, Lippincott-Schwartz J (2006) Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization. Nat Methods 3: 205–210. doi:10.1038/nmeth857. PubMed: 16489338.
[19]
Xiang Y, Wang Y (2010) GRASP55 and GRASP65 play complementary and essential roles in Golgi cisternal stacking. J Cell Biol 188: 237–251. doi:10.1083/jcb.200907132. PubMed: 20083603.
[20]
Sun Y, Shestakova A, Hunt L, Sehgal S, Lupashin V et al. (2007) Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis. Mol Biol Cell 18: 4129–4142. doi:10.1091/mbc.E07-01-0080. PubMed: 17699596.
[21]
Vasudevan C, Han W, Tan Y, Nie Y, Li D et al. (1998) The distribution and translocation of the G protein ADP-ribosylation factor 1 in live cells is determined by its GTPase activity. Journal of Cell Science 111 ( 9): 1277–1285.
[22]
Presley JF, Ward TH, Pfeifer AC, Siggia ED, Phair RD et al. (2002) Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport. Nature 417: 187–193. doi:10.1038/417187a. PubMed: 12000962.
[23]
Dimitrov A, Paupe V, Gueudry C, Sibarita J-B, Raposo G et al. (2009) The gene responsible for Dyggve-Melchior-Clausen syndrome encodes a novel peripheral membrane protein dynamically associated with the Golgi apparatus. Hum Mol Genet 18: 440–453. doi:10.1093/hmg/ddn371. PubMed: 18996921.
[24]
Linstedt AD, Mehta A, Suhan J, Reggio H, Hauri HP (1997) Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein. Mol Biol Cell 8: 1073–1087. doi:10.1091/mbc.8.6.1073. PubMed: 9201717.
[25]
Bachert C, Lee TH, Linstedt AD (2001) Lumenal endosomal and Golgi-retrieval determinants involved in pH-sensitive targeting of an early Golgi protein. Mol Biol Cell 12: 3152–3160. doi:10.1091/mbc.12.10.3152. PubMed: 11598199.
[26]
Starr T, Forsten-Williams K, Storrie B (2007) Both post-Golgi and intra-Golgi cycling affect the distribution of the Golgi phosphoprotein GPP130. Traffic 8: 1265–1279. doi:10.1111/j.1600-0854.2007.00607.x. PubMed: 17605763.
[27]
White J, Johannes L, Mallard F, Girod A, Grill S et al. (1999) Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells. J Cell Biol 147: 743–760. doi:10.1083/jcb.147.4.743. PubMed: 10562278.
[28]
Goud B, Zahraoui A, Tavitian A, Saraste J (1990) Small GTP-binding protein associated with Golgi cisternae. Nature 345: 553–556. doi:10.1038/345553a0. PubMed: 2112230.
[29]
Antony C, Cibert C, Géraud G, Santa Maria A, Maro B et al. (1992) The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach. J Cell Sci 103 ( 3): 785–796. PubMed: 1478971.
[30]
Martinez O, Antony C, Pehau-Arnaudet G, Berger EG, Salamero J et al. (1997) GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum. Proc Natl Acad Sci U S A 94: 1828–1833. doi:10.1073/pnas.94.5.1828. PubMed: 9050864.
[31]
Mallard F, Tang BL, Galli T, Tenza D, Saint-Pol A et al. (2002) Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. J Cell Biol 156: 653–664. doi:10.1083/jcb.200110081. PubMed: 11839770.
[32]
Girod A, Storrie B, Simpson JC, Johannes L, Goud B et al. (1999) Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum. Nat Cell Biol 1: 423–430. doi:10.1038/15658. PubMed: 10559986.
[33]
Del Nery E, Miserey-Lenkei S, Falguières T, Nizak C, Johannes L et al. (2006) Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking. Traffic 7: 394–407. doi:10.1111/j.1600-0854.2006.00395.x. PubMed: 16536738.
[34]
Grigoriev I, Splinter D, Keijzer N, Wulf PS, Demmers J et al. (2007) Rab6 Regulates Transport and Targeting of Exocytotic Carriers. Dev Cell 13: 305–314. doi:10.1016/j.devcel.2007.06.010. PubMed: 17681140.
[35]
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal. Omega - Mol Syst Biol 7: 539. doi:10.1038/msb.2011.75.
[36]
Gaudin R, de Alencar BC, Jouve M, Bèrre S, Le Bouder E et al. (2012) Critical role for the kinesin KIF3A in the HIV life cycle in primary human macrophages. J Cell Biol 199: 467–479. doi:10.1083/jcb.201201144. PubMed: 23091068.
[37]
Poser I, Sarov M, Hutchins JRA, Hériché J-K, Toyoda Y et al. (2008) BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 5: 409–415. doi:10.1038/nmeth.1199. PubMed: 18391959.
[38]
Moutel S, Marjou El A, Vielemeyer O, Nizak C, Benaroch P et al. (2009) A multi-Fc-species system for recombinant antibody production. BMC Biotechnol 9: 14. doi:10.1186/1472-6750-9-14. PubMed: 19245715.
[39]
Izeddin I, Boulanger J, Racine V, Specht CG, Kechkar A et al. (2012) Wavelet analysis for single molecule localization microscopy. Opt Express 20: 2081–2095. doi:10.1364/OE.20.002081. PubMed: 22330449.
[40]
Kechkar A, Nair D, Heilemann M, Choquet D, Sibarita J-B (2013) Real-time analysis and visualization for single-molecule based super-resolution microscopy. PLOS ONE 8: e62918. doi:10.1371/journal.pone.0062918. PubMed: 23646160.
[41]
Nizak C, Sougrat R, Jollivet F, Rambourg A, Goud B et al. (2004) Golgi inheritance under a block of anterograde and retrograde traffic. Traffic 5: 284–299. doi:10.1111/j.1398-9219.2004.0174.x. PubMed: 15030570.
[42]
Mallard F, Antony C, Tenza D, Salamero J, Goud B et al. (1998) Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport. J Cell Biol 143: 973–990. doi:10.1083/jcb.143.4.973. PubMed: 9817755.
[43]
Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJ et al. (1997) ER-to-Golgi transport visualized in living cells. Nature 389: 81–85. doi:10.1038/38001. PubMed: 9288971.
[44]
Boulanger J, Kervrann C, Bouthemy P (2007) Space-time adaptation for patch-based image sequence restoration. IEEE Trans Pattern Anal Mach. Intell 29: 1096–1102. doi:10.1109/TPAMI.2007.1064.
[45]
Boulanger J, Kervrann C, Bouthemy P, Elbau P, Sibarita J-B et al. (2010) Patch-based nonlocal functional for denoising fluorescence microscopy image sequences. IEEE Trans Med Imaging 29: 442–454. doi:10.1109/TMI.2009.2033991. PubMed: 19900849.
