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2011 | 58 | 4 | 597-607
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Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote

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Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala140 in Rab7a for Ser140 in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.
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  • Nencki Institute of Experimental Biology, Warszawa, Poland
  • Nencki Institute of Experimental Biology, Warszawa, Poland
  • Nencki Institute of Experimental Biology, Warszawa, Poland
  • Nencki Institute of Experimental Biology, Warszawa, Poland
  • Nencki Institute of Experimental Biology, Warszawa, Poland
  • Nencki Institute of Experimental Biology, Warszawa, Poland
  • Ali BR, Seabra MC (2005) Targeting of Rab GTPases to cellular membranes. Biochem Soc Trans 33: 652-656.
  • Ali BR, Wasmeier C, Lamoreux L, Strom M, Seabra MC (2004) Multiple regions contribute to membrane targeting of Rab GTPases. J Cell Sci 117: 6401-6412.
  • Allen RD (1974) Food vacuole membrane growth with microtubule-associated membrane transport in Paramecium. J Cell Biol 63: 904-922.
  • Allen RD, Fok AK (2000) Membrane trafficking and processing in Paramecium. Int Rev Cytol 198: 277-318.
  • Amoutzias GD, He Y, Gordon J, Mossialos D, Oliver SG, Van de Peer Y (2010) Posttranslational regulation impacts the fate of duplicated genes. Proc Natl Acad Sci USA 107: 2967-2971.
  • Andersen AP, Wyroba E, Reichman M, Zhao H, Satir BH (1994) The activity of parafusin is distinct from that of phosphoglucomutase in the unicellular eukaryote Paramecium. Biochem Biophys Res Commun 200: 1353-1358.
  • Aury JM, Jaillon O, Duret L, Noel B, Jubin C, Porcel BM, Ségurens B, Daubin V, Anthouard V, Aiach N, Arnaiz O, Billaut A, Beisson J, Blanc I, Bouhouche K, Câmara F, Duharcourt S, Guigo R, Gogendeau D, Katinka M, Keller AM, Kissmehl R, Klotz C, Koll F, Le Mouël A, Lepère G, Malinsky S, Nowacki M, Nowak JK, Plattner H, Poulain J, Ruiz F, Serrano V, Zagulski M, Dessen P, Bétermier M, Weissenbach J, Scarpelli C, Schächter V, Sperling L, Meyer E, Cohen J, Wincker P (2006) Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444: 171-178.
  • Axelsen BJ, Yan KK, Maslov S (2007) Parameters of proteome evolution from histograms of amino-acid sequence identities of paralogous proteins. Biol Direct 2: 32.
  • Bierczynska-Krzysik A, Bonar E, Drabik A, Noga M, Suder P, Dylag T, Dubin A, Kotlinska J, Silberring J (2006) Rat brain proteome in morphine dependence. Neurochem Int 49: 401-406.
  • Brighouse A, Dacks JB, Field MC (2010) Rab protein evolution and the history of the eukaryotic endomembrane system. Cell Mol Life Sci 67: 3449-3465.
  • Bright LJ, Kambesis N, Nelson SB, Jeong B, Turkewitz AP (2010) Comprehensive analysis reveals dynamic and evolutionary plasticity of Rab GTPases and membrane traffic in Tetrahymena thermophila. PLoS Genet 6: e1001155.
  • Bucci C, Chiariello M (2006) Signal transduction gRABs attention. Cell Signal 18: 1-8.
  • Bucci C, Thomsen P, Nicoziani P, McCarthy J, van Deurs B (2000) Rab7: a key to lysosome biogenesis. Mol Biol Cell 11: 467-480.
  • Clerot J, Iftode F, Budin K, Jeanmaire-Wolf R, Coffe G, Fleury-Aubusson A (2001) Fine oral filaments in Paramecium: a biochemical and immunological analysis. J Eukaryot Microbiol 48: 234-245.
  • Cohen J, Beisson J (1988) The Cytoskeleton. In Paramecium. Görtz HD, ed, pp 363-392. Springer-Verlag, Berlin.
  • Dacks JB, Poon PP, Field MC (2008) Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution. Proc Natl Acad Sci USA 105: 588-593.
  • Damaj R, Pomel S, Bricheux G, Coffe G, Viguès B, Ravet V, Bouchard P (2009) Cross-study analysis of genomic data defines the ciliate multigenic epiplasmin family: strategies for functional analysis in Paramecium tetraurelia. BMC Evol Biol 9: 125.
  • Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, Wortman JR, Badger JH, Ren Q, Amedeo P, Jones KM, Tallon LJ, Delcher AL, Salzberg SL, Silva JC, Haas BJ, Majoros WH, Farzad M, Carlton JM, Smith RK Jr, Garg J, Pearlman RE, Karrer KM, Sun L, Manning G, Elde NC, Turkewitz AP, Asai DJ, Wilkes DE, Wang Y, Cai H, Collins K, Stewart BA, Lee SR, Wilamowska K, Weinberg Z, Ruzzo WL, Wloga D, Gaertig J, Frankel J, Tsao CC, Gorovsky MA, Keeling PJ, Waller RF, Patron NJ, Cherry JM, Stover NA, Krieger CJ, del Toro C, Ryder HF, Williamson SC, Barbeau RA, Hamilton EP, Orias E (2006) Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol 4: e286.
  • Elias M (2010) Patterns and processes in the evolution of the eukaryotic endomembrane system. Mol Membr Biol 27: 469-489.
  • Galvani A, Sperling L (2002) RNA interference by feeding in Paramecium. Trends Genet 18: 11-12.
  • Gunther GR, Wang JL, Yahara I, Cunningham BA, Edelman GM (1973) Concanavalin A derivatives with altered biological activities. Proc Natl Acad Sci USA 70: 1012-1016.
  • Ho MN, Hirata R, Umemoto N, Ohya Y, Takatsuki A, Stevens TH, Anraku Y (1993) VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae. J Biol Chem 268: 18286-18292.
  • Iftode F, Clérot JC, Levilliers N, Bré MH (2000) Tubulin polyglycylation: a morphogenetic marker in ciliates. Biol Cell 92: 615-628.
  • Itzen A, Goody RS (2010) GTPases involved in vesicular trafficking: structures and mechanisms. Semin Cell Dev Biol 22: 48-56.
  • Jékely G (2007) Origin of eukaryotic endomembranes: a critical evaluation of different model scenarios. Adv Exp Med Biol 607: 38-51.
  • Julenius K, Mølgaard A, Gupta R, Brunak S (2005) Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15: 153-164.
  • Kashiwazaki J, Iwaki T, Takegawa K, Shimoda C, Nakamura T (2009) Two fission yeast Rab7 homologs, Ypt7 and Ypt71, play antagonistic roles in the regulation of vacuolar morphology. Traffic 10: 912-924.
  • Kinchen JM, Ravichandran KS (2008) Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol 9: 781-795.
  • Kissmehl R, Schilde C, Wassmer T, Danzer C, Nuehse K, Lutter K, Plattner H (2007) Molecular identification of 26 syntaxin genes and their assignment to the different trafficking pathways in Paramecium. Traffic 8: 523-542.
  • Klotz C, Garreau de Loubresse N, Ruiz F, Beisson J (1997) Genetic evidence for a role of centrin-associated proteins in the organization and dynamics of the infraciliary lattice in Paramecium. Cell Motil Cytoskeleton 38: 172-86.
  • Lee MT, Mishra A, Lambright DG (2009) Structural mechanisms for regulation of membrane traffic by rab GTPases. Traffic 10: 1377-1389.
  • Lewis UJ, Singh RN, Sinha YN, Vanderlaan WP (1985) Glycosylated human prolactin. Endocrinology 116: 359-363.
  • Liu T, Mirschberger C, Chooback L, Arana Q, Dal Sacco Z, MacWilliams H, Clarke M (2002) Altered expression of the 100 kDa subunit of the Dictyostelium vacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation. J Cell Sci 115: 1907-1918.
  • Liu L, Wyroba E, Satir BH (2011) RNAi Knockdown of Parafusin Inhibits The Secretory Pathway. Eur J Cell Biol 90: 844-853.
  • Mackiewicz P, Wyroba E (2009) Phylogeny and evolution of Rab7 and Rab9 proteins. BMC Evol Biol 9: 101.
  • Matthiesen SH, Shenoy SM, Kim K, Singer RH, Satir BH (2001) A parafusin-related Toxoplasma protein in Ca2+ regulated secretory organelles. Eur J Cell Biol 80: 775-783.
  • Noga MT, Lewandowski JJ, Suder P, Silberring J (2005) An enhanced method for peptides sequencing by N-terminal derivatization and MS. Proteomics 5: 4367-4375.
  • Okada M, Huston CD, Mann BJ, Petri WA Jr, Kita K, Nozaki T (2005) Proteomic analysis of phagocytosis in the enteric protozoan parasite Entamoeba histolytica. Eukaryot Cell 4: 827-831.
  • Parra KJ, Keenan KL, Kane PM (2000) The H subunit (Vma13p) of the yeast ATPase inhibits the ATPase activity of cytosolic V1 complexes. J Biol Chem 275: 21761-21767.
  • Pfeffer SR, Aivazian D (2004) Targeting Rab GTPases to distinct membrane compartments. Nat Rev Mol Cell Biol 5: 886-896.
  • Plattner H (2010) Membrane trafficking in protozoa SNARE proteins, H+-ATPase, actin, and other key players in ciliates. Int Rev Cell Mol Biol 280: 79-184.
  • Price AE, Logvinenko KB, Higgins EA, Cole ES, Richards S (1995) Studies on the microheterogeneity and in vitro activity of glycosylated and nonglycosylated recombinant human prolactin separated using a novel purification process. Endocrinology 136: 4827-4833.
  • Progida C, Cogli L, Piro F, De Luca A, Bakke O, Bucci C (2010) Rab7b controls trafficking from endosomes to the TGN. J Cell Sci 123: 1480-1491.
  • Rupper A, Grove B, Cardelli J (2001) Rab7 regulates phagosome maturation in Dictyostelium. J Cell Sci 114: 2449-2460.
  • Saito-Nakano Y, Loftus BJ, Hall N, Nozaki T (2005) The diversity of Rab GTPases in Entamoeba histolytica. Exp Parasitol 110: 244-252.
  • Saito-Nakano Y, Nakahara T, Nakano K, Nozaki T, Numata O (2010) Marked amplification and diversification of products of ras genes from rat brain, Rab GTPases, in the ciliates Tetrahymena thermophila and Paramecium tetraurelia. J Eukaryot Microbiol 57: 389 - 399.
  • Satir BH, Srisomsap C, Reichman M, Marchase RB (1990) Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver. J Cell Biol 111: 901-917.
  • Sehring IM, Reiner C, Mansfeld J, Plattner H, Kissmehl R (2007) A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function. J Cell Sci 120: 177-190.
  • Sillo A, Bloomfield G, Balest A, Balbo A, Pergolizzi B, Peracino B, Skelton J, Ivens A, Bozzaro S (2008) Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium. BMC Genomics 9: 291.
  • Soldo AT, Godoy GA, van Wagtendonk WJ (1966) Growth of particle-bearing and particle-free Paramecium aurelia in axenic culture. J Protozool 13: 492-497.
  • Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10: 513-525.
  • Stenmark H, Olkkonen VM (2001) The Rab GTPase family. Genome Biol 2: reviews 3007.1-3007.7
  • Subramanian SV, Satir BH (1992) Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca2+-dependent transducer molecule in exocytosis in Paramecium. Proc Natl Acad Sci USA 89: 11297-11301.
  • Subramanian SV, Wyroba E, Andersen AP, Satir BH (1994) Cloning and sequencing of parafusin, a calcium-dependent exocytosis-related phosphoglycoprotein. Proc Natl Acad Sci USA 91: 9832-9836.
  • Surmacz L, Wiejak J, Wyroba E (2003) Evolutionary conservancy of the endocytic machinery in the unicellular eukaryote Paramecium. Biol Cell 95: 69-74.
  • Surmacz L, Wiejak J, Wyroba E (2006) Cloning of two genes encoding Rab7 in Paramecium. Acta Biochim Pol 53: 149-156.
  • Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395: 854.
  • VanderSluis B, Bellay J, Musso G, Costanzo M, Papp B, Vizeacoumar FJ, Baryshnikova A, Andrews B, Boone C, Myers CL (2010) Genetic interactions reveal the evolutionary trajectories of duplicate genes. Mol Syst Biol 6: 429.
  • Vanlandingham PA, Ceresa BP (2009) Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration. J Biol Chem 284: 12110-12124.
  • Vieira OV, Botelho RJ, Grinstein S (2002) Phagosome maturation: aging gracefully. Biochem J 366: 689-704.
  • Wassmer T, Sehring IM, Kissmehl R, Plattner H (2009) The V-ATPase In Paramecium: functional specialization by multiple gene isoforms. Pflugers Arch 457: 599-607.
  • Wiejak J, Surmacz L, Wyroba E (2004) Dynamin- and clathrin-dependent endocytic pathway in unicellular eukaryote Paramecium. Biochem Cell Biol 82: 547-558.
  • Winer J, Jung CK, Shackel I, Williams PM (1999) Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270: 41-49.
  • Wyroba E, Surmacz L, Osinska M, Wiejak J (2007) Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues. Eur J Histochem 51: 163-172.
  • Yaqoob M, Snell CR, Burgess GM (1995) Carbohydrate analysis of the B2 bradykinin receptor from rat uterus. J Neurochem 65: 1290-1296.
  • Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2: 107-117.
  • Zimmerman JM, Eliezer N, Simha R (1968) The characterization of amino acid sequences in proteins by statistical methods. J Theor Biol 21: 170-201.
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