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Planta

, Volume 245, Issue 4, pp 779–792 | Cite as

Localization of RNS2 ribonuclease to the vacuole is required for its role in cellular homeostasis

  • Brice E. Floyd
  • Yosia Mugume
  • Stephanie C. Morriss
  • Gustavo C. MacIntoshEmail author
  • Diane C. BasshamEmail author
Original Article

Abstract

Main conclusion

Localization of the RNase RNS2 to the vacuole via a C-terminal targeting signal is essential for its function in rRNA degradation and homeostasis.

RNase T2 ribonucleases are highly conserved enzymes present in the genomes of nearly all eukaryotes and many microorganisms. Their constitutive expression in different tissues and cell types of many organisms suggests a housekeeping role in RNA homeostasis. The Arabidopsis thaliana class II RNase T2, RNS2, is encoded by a single gene and functions in rRNA degradation. Loss of RNS2 results in RNA accumulation and constitutive activation of autophagy, possibly as a compensatory mechanism. While the majority of RNase T2 enzymes is secreted, RNS2 is located within the vacuole and in the endoplasmic reticulum (ER), possibly within ER bodies. As RNS2 has a neutral pH optimum, and the endomembrane organelles are connected by vesicle transport, the site within the endomembrane system at which RNS2 functions is unclear. Here we demonstrate that localization to the vacuole is essential for the physiological function of RNS2. A mutant allele of RNS2, rns2-1, results in production of an active RNS2 RNase but with a mutation that removes a putative C-terminal vacuolar targeting signal. The mutant protein is, therefore, secreted from the cell. This results in a constitutive autophagy phenotype similar to that observed in rns2 null mutants. These findings illustrate that the intracellular retention of RNS2 and localization within the vacuole are critical for its cellular function.

Keywords

Arabidopsis Autophagy Ribosomal RNA RNA degradation Vacuolar targeting 

Abbreviations

ConcA

Concanamycin A

ER

Endoplasmic reticulum

MDC

Monodansylcadaverine

WT

Wild-type

Notes

Acknowledgements

This work was supported by Grant No. MCB-1051818 from the United States National Science Foundation to GCM and DCB and Grant No. DE-SC0014038 from the United States Department of Energy to DCB. We thank Danielle Ebany for isolation of an rns2-1 homozygote and Junmarie Soto-Burgos for assistance with confocal microscopy.

Supplementary material

425_2016_2644_MOESM1_ESM.pdf (793 kb)
Supplementary material 1 (PDF 793 kb)

References

  1. Ahmed SU, Rojo E, Kovaleva V, Venkataraman S, Dombrowski JE, Matsuoka K, Raikhel NV (2000) The plant vacuolar sorting receptor AtELP is involved in transport of NH2-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana. J Cell Biol 149:1335–1344. doi: 10.1083/jcb.149.7.1335 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Andersen KL, Collins K (2012) Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena. Mol Biol Cell 23(1):36–44. doi: 10.1091/mbc.E11-08-0689 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Andersen KR, Jensen TH, Brodersen DE (2008) Take the “A” tail–quality control of ribosomal and transfer RNA. Biochim Biophys Acta 1779(9):532–537. doi: 10.1016/j.bbagrm.2008.06.011 CrossRefPubMedGoogle Scholar
  4. Balagopal V, Parker R (2009) Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21(3):403–408. doi: 10.1016/j.ceb.2009.03.005 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bariola PA, Howard CJ, Taylor CB, Verburg MT, Jaglan VD, Green PJ (1994) The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J 6(5):673–685CrossRefPubMedGoogle Scholar
  6. Bariola PA, MacIntosh GC, Green PJ (1999) Regulation of S-like ribonuclease levels in Arabidopsis. Antisense inhibition of RNS1 or RNS2 elevates anthocyanin accumulation. Plant Physiol 119(1):331–342CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bednarek SY, Wilkins TA, Dombrowski JE, Raikhel NV (1990) A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell 2(12):1145–1155. doi: 10.1105/tpc.2.12.1145 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Carter C, Pan S, Zouhar J, Avila E, Girke T, Raikhel N (2004) The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16(12):3285–3303CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cervelli M, Di Caro O, Di Penta A, Angelini R, Federico R, Vitale A, Mariottini P (2004) A novel C-terminal sequence from barley polyamine oxidase is a vacuolar sorting signal. Plant J 40(3):410–418. doi: 10.1111/j.1365-313X.2004.02221.x CrossRefPubMedGoogle Scholar
  10. Chou KC, Shen HB (2010) Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS One 5(6):e11335. doi: 10.1371/journal.pone.0011335 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Clough S, Bent A (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743CrossRefPubMedGoogle Scholar
  12. Condon C, Putzer H (2002) The phylogenetic distribution of bacterial ribonucleases. Nucleic Acids Res 30(24):5339–5346CrossRefPubMedPubMedCentralGoogle Scholar
  13. Contento AL, Xiong Y, Bassham DC (2005) Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein. Plant J 42(4):598–608CrossRefPubMedGoogle Scholar
  14. Dettmer J, Hong-Hermesdorf A, Stierhof YD, Schumacher K (2006) Vacuolar H+ -ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18(3):715–730. doi: 10.1105/tpc.105.037978 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Drose S, Bindseil KU, Bowman EJ, Siebers A, Zeeck A, Altendorf K (1993) Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32(15):3902–3906CrossRefPubMedGoogle Scholar
  16. Dyer KD, Rosenberg HF (2006) The RNase A superfamily: generation of diversity and innate host defense. Mol Divers 10(4):585–597. doi: 10.1007/s11030-006-9028-2 CrossRefPubMedGoogle Scholar
  17. Einhauer A, Jungbauer A (2001) The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods 49(1–3):455–465CrossRefPubMedGoogle Scholar
  18. Floyd BE, Morriss SC, Macintosh GC, Bassham DC (2012) What to eat: evidence for selective autophagy in plants. J Integr Plant Biol 54(11):907–920. doi: 10.1111/j.1744-7909.2012.01178.x PubMedGoogle Scholar
  19. Floyd BE, Morriss SC, MacIntosh GC, Bassham DC (2015) Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis. Autophagy 11(12):2199–2212. doi: 10.1080/15548627.2015.1106664 CrossRefPubMedGoogle Scholar
  20. Fuji K, Shirakawa M, Shimono Y, Kunieda T, Fukao Y, Koumoto Y, Takahashi H, Hara-Nishimura I, Shimada T (2016) The adaptor complex AP-4 regulates vacuolar protein sorting at the trans-Golgi network by interacting with VACUOLAR SORTING RECEPTOR1. Plant Physiol 170(1):211–219. doi: 10.1104/pp.15.00869 CrossRefPubMedGoogle Scholar
  21. Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y (2002) Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol 129(3):1181–1193CrossRefPubMedPubMedCentralGoogle Scholar
  22. Haud N, Kara F, Diekmann S, Henneke M, Willer JR, Hillwig MS, Gregg RG, Macintosh GC, Gartner J, Alia A, Hurlstone AF (2011) rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for RNase T2 in degrading ribosomal RNA. Proc Natl Acad Sci USA 108(3):1099–1103. doi: 10.1073/pnas.1009811107 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Henneke M, Diekmann S, Ohlenbusch A, Kaiser J, Engelbrecht V, Kohlschutter A, Kratzner R, Madruga-Garrido M, Mayer M, Opitz L, Rodriguez D, Ruschendorf F, Schumacher J, Thiele H, Thoms S, Steinfeld R, Nurnberg P, Gartner J (2009) RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection. Nat Genet 41(7):773–775. doi: 10.1038/ng.398 CrossRefPubMedGoogle Scholar
  24. Hillwig MS, Rizhsky L, Wang Y, Umanskaya A, Essner JJ, MacIntosh GC (2009) Zebrafish RNase T2 genes and the evolution of secretory ribonucleases in animals. BMC Evol Biol 9:170. doi: 10.1186/1471-2148-9-170 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hillwig MS, Contento AL, Meyer A, Ebany D, Bassham DC, Macintosh GC (2011) RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants. Proc Natl Acad Sci USA 108(3):1093–1098. doi: 10.1073/pnas.1009809108 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Houseley J, Tollervey D (2009) The many pathways of RNA degradation. Cell 136(4):763–776. doi: 10.1016/j.cell.2009.01.019 CrossRefPubMedGoogle Scholar
  27. Hua ZH, Fields A, Kao TH (2008) Biochemical models for S-RNase-based self-incompatibility. Mol Plant 1(4):575–585. doi: 10.1093/mp/ssn032 CrossRefPubMedGoogle Scholar
  28. Huang H, Kawamata T, Horie T, Tsugawa H, Nakayama Y, Ohsumi Y, Fukusaki E (2015) Bulk RNA degradation by nitrogen starvation-induced autophagy in yeast. EMBO J 34(2):154–168. doi: 10.15252/embj.201489083 CrossRefPubMedGoogle Scholar
  29. Hugot K, Ponchet M, Marais A, Ricci P, Galiana E (2002) A tobacco S-like RNase inhibits hyphal elongation of plant pathogens. Mol Plant Microbe Interact 15(3):243–250. doi: 10.1094/mpmi.2002.15.3.243 CrossRefPubMedGoogle Scholar
  30. Igic B, Kohn JR (2001) Evolutionary relationships among self-incompatibility RNases. Proc Natl Acad Sci USA 98(23):13167–13171. doi: 10.1073/pnas.231386798 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Irie M (1999) Structure-function relationships of acid ribonucleases: lysosomal, vacuolar, and periplasmic enzymes. Pharmacol Ther 81(2):77–89CrossRefPubMedGoogle Scholar
  32. Jost W, Bak H, Glund K, Terpstra P, Beintema JJ (1991) Amino acid sequence of an extracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur J Biochem 198(1):1–6CrossRefPubMedGoogle Scholar
  33. Kang H, Kim SY, Song K, Sohn EJ, Lee Y, Lee DW, Hara-Nishimura I, Hwang I (2012) Trafficking of vacuolar proteins: the crucial role of Arabidopsis vacuolar protein sorting 29 in recycling vacuolar sorting receptor. Plant Cell 24(12):5058–5073. doi: 10.1105/tpc.112.103481 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Koide Y, Matsuoka K, Ohto M, Nakamura K (1999) The N-terminal propeptide and the C terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of vacuolar sorting signals. Plant Cell Physiol 40(11):1152–1159CrossRefPubMedGoogle Scholar
  35. Kothke S, Kock M (2011) The Solanum lycopersicum RNaseLER is a class II enzyme of the RNase T2 family and shows preferential expression in guard cells. J Plant Physiol 168(8):840–847. doi: 10.1016/j.jplph.2010.11.012 CrossRefPubMedGoogle Scholar
  36. Kraft C, Deplazes A, Sohrmann M, Peter M (2008) Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol 10(5):602–610. doi: 10.1038/ncb1723 CrossRefPubMedGoogle Scholar
  37. Kurata N, Kariu T, Kawano S, Kimura M (2002) Molecular cloning of cDNAs encoding ribonuclease-related proteins in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection. Biosci Biotechnol Biochem 66(2):391–397. doi: 10.1271/bbb.66.391 CrossRefPubMedGoogle Scholar
  38. Lehmann K, Hause B, Altmann D, Kock M (2001) Tomato ribonuclease LX with the functional endoplasmic reticulum retention motif HDEF is expressed during programmed cell death processes, including xylem differentiation, germination, and senescence. Plant Physiol 127(2):436–449CrossRefPubMedPubMedCentralGoogle Scholar
  39. Liu Y, Burgos JS, Deng Y, Srivastava R, Howell SH, Bassham DC (2012) Degradation of the endoplasmic reticulum by autophagy during endoplasmic reticulum stress in Arabidopsis. Plant Cell 24(11):4635–4651. doi: 10.1105/tpc.112.101535 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Loffler A, Abel S, Jost W, Beintema JJ, Glund K (1992) Phosphate-regulated induction of intracellular ribonucleases in cultured tomato (Lycopersicon esculentum) cells. Plant Physiol 98(4):1472–1478CrossRefPubMedPubMedCentralGoogle Scholar
  41. MacIntosh GC (2011) RNase T2 Family: Enzymatic properties, functional diversity, and evolution of ancient ribonucleases. In: Nicholson AWW (ed) ribonucleases, vol 26. Springer, Berlin Heidelberg, pp 89–114CrossRefGoogle Scholar
  42. MacIntosh GC, Bariola PA, Newbigin E, Green PJ (2001) Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes? Proc Natl Acad Sci USA 98(3):1018–1023. doi: 10.1073/pnas.98.3.1018 CrossRefPubMedPubMedCentralGoogle Scholar
  43. MacIntosh GC, Hillwig MS, Meyer A, Flagel L (2010) RNase T2 genes from rice and the evolution of secretory ribonucleases in plants. Mol Genet Genomics 283(4):381–396. doi: 10.1007/s00438-010-0524-9 CrossRefPubMedGoogle Scholar
  44. Matsushima R, Hayashi Y, Kondo M, Shimada T, Nishimura M, Hara-Nishimura I (2002) An endoplasmic reticulum-derived structure that is induced under stress conditions in Arabidopsis. Plant Physiol 130(4):1807–1814. doi: 10.1104/pp.009464 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Meng X, Sun P, Kao TH (2011) S-RNase-based self-incompatibility in Petunia inflata. Ann Bot 108(4):637–646. doi: 10.1093/aob/mcq253 CrossRefPubMedGoogle Scholar
  46. Nakano RT, Yamada K, Bednarek P, Nishimura M, Hara-Nishimura I (2014) ER bodies in plants of the Brassicales order: biogenesis and association with innate immunity. Front Plant Sci 5:73. doi: 10.3389/fpls.2014.00073 PubMedPubMedCentralGoogle Scholar
  47. Neuhaus JM, Sticher L, Meins F Jr, Boller T (1991) A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88(22):10362–10366CrossRefPubMedPubMedCentralGoogle Scholar
  48. Niemes S, Labs M, Scheuring D, Krueger F, Langhans M, Jesenofsky B, Robinson DG, Pimpl P (2010a) Sorting of plant vacuolar proteins is initiated in the ER. Plant J 62(4):601–614CrossRefPubMedGoogle Scholar
  49. Niemes S, Langhans M, Viotti C, Scheuring D, San Wan Yan M, Jiang L, Hillmer S, Robinson DG, Pimpl P (2010b) Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J 61(1):107–121. doi: 10.1111/j.1365-313X.2009.04034.x CrossRefPubMedGoogle Scholar
  50. Noda T, Kim J, Huang WP, Baba M, Tokunaga C, Ohsumi Y, Klionsky DJ (2000) Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. J Cell Biol 148(3):465–480CrossRefPubMedPubMedCentralGoogle Scholar
  51. Okabe T, Yoshimoto I, Hitoshi M, Ogawa T, Ohyama T (2005) An S-like ribonuclease gene is used to generate a trap-leaf enzyme in the carnivorous plant Drosera adelae. FEBS Lett 579(25):5729–5733. doi: 10.1016/j.febslet.2005.09.043 CrossRefPubMedGoogle Scholar
  52. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10):785–786. doi: 10.1038/nmeth.1701 CrossRefPubMedGoogle Scholar
  53. Robert S, Zouhar J, Carter C, Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts. Nat Protoc 2(2):259–262CrossRefPubMedGoogle Scholar
  54. Rojas H, Floyd B, Morriss S, Bassham D, MacIntosh G, Goldraij A (2015) NnSR1, a class III non-S-RNase specifically induced in Nicotiana alata under Pi deficiency, is localized in endoplasmic reticulum compartments. Plant Sci 236:250–259CrossRefPubMedGoogle Scholar
  55. Saalbach G, Rosso M, Schumann U (1996) The vacuolar targeting signal of the 2S albumin from Brazil nut resides at the C terminus and involves the C-terminal propeptide as an essential element. Plant Physiol 112(3):975–985CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sanderfoot A, Ahmed S, Marty-Mazars D, Rapoport I, Kirchhausen T, Marty F, Raikhel N (1998) A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots. Proc Natl Acad Sci USA 95(17):9920–9925CrossRefPubMedPubMedCentralGoogle Scholar
  57. Sanderfoot AA, Kovaleva V, Bassham DC, Raikhel NV (2001) Interactions between syntaxins identify at least five SNARE complexes within the Golgi/prevacuolar system of the Arabidopsis cell. Mol Biol Cell 12(12):3733–3743CrossRefPubMedPubMedCentralGoogle Scholar
  58. Sheen J (2002) A transient expression assay using Arabidopsis mesophyll protoplasts. Available online at http://molbio.mgh.harvard.edu/sheenweb/protocols_reg.html
  59. Tapernoux-Luthi EM, Schneider T, Keller F (2007) The C-terminal sequence from common bugle leaf galactan:galactan galactosyltransferase is a non-sequence-specific vacuolar sorting determinant. FEBS Lett 581(9):1811–1818. doi: 10.1016/j.febslet.2007.03.068 CrossRefPubMedGoogle Scholar
  60. Taylor CB, Green PJ (1991) Genes with homology to fungal and S-Gene RNases are expressed in Arabidopsis thaliana. Plant Physiol 96(3):980–984CrossRefPubMedPubMedCentralGoogle Scholar
  61. Taylor CB, Bariola PA, delCardayre SB, Raines RT, Green PJ (1993) RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc Natl Acad Sci USA 90(11):5118–5122CrossRefPubMedPubMedCentralGoogle Scholar
  62. Vitale A, Denecke J (1999) The endoplasmic reticulum-gateway of the secretory pathway. Plant Cell 11(4):615–628PubMedPubMedCentralGoogle Scholar
  63. Webber JL, Tooze SA (2010) New insights into the function of Atg9. FEBS Lett 584(7):1319–1326. doi: 10.1016/j.febslet.2010.01.020 CrossRefPubMedGoogle Scholar
  64. Yamamoto H, Kakuta S, Watanabe TM, Kitamura A, Sekito T, Kondo-Kakuta C, Ichikawa R, Kinjo M, Ohsumi Y (2012) Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 198(2):219–233. doi: 10.1083/jcb.201202061 CrossRefPubMedPubMedCentralGoogle Scholar
  65. Yang X, Bassham DC (2015) New insight into the mechanism and function of autophagy in plant cells. Int Rev Cell Mol Biol 320:1–40. doi: 10.1016/bs.ircmb.2015.07.005 CrossRefPubMedGoogle Scholar
  66. Ye ZH, Droste DL (1996) Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans. Plant Mol Biol 30(4):697–709CrossRefPubMedGoogle Scholar
  67. Yen Y, Green PJ (1991) Identification and properties of the major ribonucleases of Arabidopsis thaliana. Plant Physiol 97(4):1487–1493CrossRefPubMedPubMedCentralGoogle Scholar
  68. Yoshida H (2001) The ribonuclease T1 family. Methods Enzymol 341:28–41CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Brice E. Floyd
    • 1
  • Yosia Mugume
    • 1
  • Stephanie C. Morriss
    • 2
  • Gustavo C. MacIntosh
    • 2
    Email author
  • Diane C. Bassham
    • 1
    Email author
  1. 1.Department of Genetics, Development and Cell BiologyIowa State UniversityAmesUSA
  2. 2.Roy J. Carver Department of Biochemistry, Biophysics and Molecular BiologyIowa State UniversityAmesUSA

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