Skip to main content
PMC home page
RNA logoLink to RNA
. 2001 Oct;7(10):1464–1475.

Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

S Baginsky 1, A Shteiman-Kotler 1, V Liveanu 1, S Yehudai-Resheff 1, M Bellaoui 1, R E Settlage 1, J Shabanowitz 1, D F Hunt 1, G Schuster 1, W Gruissem 1
PMCID: PMC1370190  PMID: 11680851

Abstract

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.

Full Text

The Full Text of this article is available as a PDF (174.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alconada A., Flores A. I., Blanco L., Cuezva J. M. Antibodies against F1-ATPase alpha-subunit recognize mitochondrial chaperones. Evidence for an evolutionary relationship between chaperonin and ATPase protein families. J Biol Chem. 1994 May 6;269(18):13670–13679. [PubMed] [Google Scholar]
  2. Baginsky S., Gruissem W. Chloroplast mRNA 3'-end nuclease complex. Methods Enzymol. 2001;342:408–419. doi: 10.1016/s0076-6879(01)42562-6. [DOI] [PubMed] [Google Scholar]
  3. Blum E., Py B., Carpousis A. J., Higgins C. F. Polyphosphate kinase is a component of the Escherichia coli RNA degradosome. Mol Microbiol. 1997 Oct;26(2):387–398. doi: 10.1046/j.1365-2958.1997.5901947.x. [DOI] [PubMed] [Google Scholar]
  4. Carpousis A. J., Van Houwe G., Ehretsmann C., Krisch H. M. Copurification of E. coli RNAase E and PNPase: evidence for a specific association between two enzymes important in RNA processing and degradation. Cell. 1994 Mar 11;76(5):889–900. doi: 10.1016/0092-8674(94)90363-8. [DOI] [PubMed] [Google Scholar]
  5. Carpousis A. J., Vanzo N. F., Raynal L. C. mRNA degradation. A tale of poly(A) and multiprotein machines. Trends Genet. 1999 Jan;15(1):24–28. doi: 10.1016/s0168-9525(98)01627-8. [DOI] [PubMed] [Google Scholar]
  6. Coburn G. A., Mackie G. A. Degradation of mRNA in Escherichia coli: an old problem with some new twists. Prog Nucleic Acid Res Mol Biol. 1999;62:55–108. doi: 10.1016/s0079-6603(08)60505-x. [DOI] [PubMed] [Google Scholar]
  7. Coburn G. A., Miao X., Briant D. J., Mackie G. A. Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase. Genes Dev. 1999 Oct 1;13(19):2594–2603. doi: 10.1101/gad.13.19.2594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen S. N., McDowall K. J. RNase E: still a wonderfully mysterious enzyme. Mol Microbiol. 1997 Mar;23(6):1099–1106. doi: 10.1111/j.1365-2958.1997.tb02593.x. [DOI] [PubMed] [Google Scholar]
  9. Cormack R. S., Genereaux J. L., Mackie G. A. RNase E activity is conferred by a single polypeptide: overexpression, purification, and properties of the ams/rne/hmp1 gene product. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9006–9010. doi: 10.1073/pnas.90.19.9006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dickson R., Weiss C., Howard R. J., Alldrick S. P., Ellis R. J., Lorimer G., Azem A., Viitanen P. V. Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding. J Biol Chem. 2000 Apr 21;275(16):11829–11835. doi: 10.1074/jbc.275.16.11829. [DOI] [PubMed] [Google Scholar]
  11. Gagliardi D., Leaver C. J. Polyadenylation accelerates the degradation of the mitochondrial mRNA associated with cytoplasmic male sterility in sunflower. EMBO J. 1999 Jul 1;18(13):3757–3766. doi: 10.1093/emboj/18.13.3757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gruissem W., Greenberg B. M., Zurawski G., Hallick R. B. Chloroplast gene expression and promoter identification in chloroplast extracts. Methods Enzymol. 1986;118:253–270. doi: 10.1016/0076-6879(86)18077-3. [DOI] [PubMed] [Google Scholar]
  13. Grunberg-Manago M. Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annu Rev Genet. 1999;33:193–227. doi: 10.1146/annurev.genet.33.1.193. [DOI] [PubMed] [Google Scholar]
  14. Hajeer A. H., Bernstein R. M. Antibody to mycobacterial 65-kD heat shock protein in commercial antisera. Clin Exp Immunol. 1993 Dec;94(3):544–547. doi: 10.1111/j.1365-2249.1993.tb08232.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hayes R., Kudla J., Gruissem W. Degrading chloroplast mRNA: the role of polyadenylation. Trends Biochem Sci. 1999 May;24(5):199–202. doi: 10.1016/s0968-0004(99)01388-2. [DOI] [PubMed] [Google Scholar]
  16. Hayes R., Kudla J., Schuster G., Gabay L., Maliga P., Gruissem W. Chloroplast mRNA 3'-end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins. EMBO J. 1996 Mar 1;15(5):1132–1141. [PMC free article] [PubMed] [Google Scholar]
  17. Kaberdin V. R., Miczak A., Jakobsen J. S., Lin-Chao S., McDowall K. J., von Gabain A. The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly. Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11637–11642. doi: 10.1073/pnas.95.20.11637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Klaff P., Gruissem W. Changes in Chloroplast mRNA Stability during Leaf Development. Plant Cell. 1991 May;3(5):517–529. doi: 10.1105/tpc.3.5.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kudla J., Hayes R., Gruissem W. Polyadenylation accelerates degradation of chloroplast mRNA. EMBO J. 1996 Dec 16;15(24):7137–7146. [PMC free article] [PubMed] [Google Scholar]
  20. Li Z., Pandit S., Deutscher M. P. RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA. EMBO J. 1999 May 17;18(10):2878–2885. doi: 10.1093/emboj/18.10.2878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liou G. G., Jane W. N., Cohen S. N., Lin N. S., Lin-Chao S. RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E. Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):63–68. doi: 10.1073/pnas.011535498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lisitsky I., Klaff P., Schuster G. Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13398–13403. doi: 10.1073/pnas.93.23.13398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lisitsky I., Kotler A., Schuster G. The mechanism of preferential degradation of polyadenylated RNA in the chloroplast. The exoribonuclease 100RNP/polynucleotide phosphorylase displays high binding affinity for poly(A) sequence. J Biol Chem. 1997 Jul 11;272(28):17648–17653. doi: 10.1074/jbc.272.28.17648. [DOI] [PubMed] [Google Scholar]
  24. Lisitsky I., Liveanu V., Schuster G. RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein. Nucleic Acids Res. 1994 Nov 11;22(22):4719–4724. doi: 10.1093/nar/22.22.4719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lisitsky I., Schuster G. Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase. Eur J Biochem. 1999 Apr;261(2):468–474. doi: 10.1046/j.1432-1327.1999.00285.x. [DOI] [PubMed] [Google Scholar]
  26. Lopez P. J., Marchand I., Joyce S. A., Dreyfus M. The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. Mol Microbiol. 1999 Jul;33(1):188–199. doi: 10.1046/j.1365-2958.1999.01465.x. [DOI] [PubMed] [Google Scholar]
  27. Lupold D. S., Caoile A. G., Stern D. B. Polyadenylation occurs at multiple sites in maize mitochondrial cox2 mRNA and is independent of editing status. Plant Cell. 1999 Aug;11(8):1565–1578. doi: 10.1105/tpc.11.8.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Margossian S. P., Li H., Zassenhaus H. P., Butow R. A. The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity. Cell. 1996 Jan 26;84(2):199–209. doi: 10.1016/s0092-8674(00)80975-7. [DOI] [PubMed] [Google Scholar]
  29. Martin S. E., Shabanowitz J., Hunt D. F., Marto J. A. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal Chem. 2000 Sep 15;72(18):4266–4274. doi: 10.1021/ac000497v. [DOI] [PubMed] [Google Scholar]
  30. Merril C. R., Goldman D., Van Keuren M. L. Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:230–239. doi: 10.1016/s0076-6879(83)96021-4. [DOI] [PubMed] [Google Scholar]
  31. Min J., Zassenhaus H. P. A nucleoside triphosphate-regulated, 3' exonucleolytic mechanism is involved in turnover of yeast mitochondrial RNAs. J Bacteriol. 1993 Oct;175(19):6245–6253. doi: 10.1128/jb.175.19.6245-6253.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mohanty B. K., Kushner S. R. Polynucleotide phosphorylase functions both as a 3' right-arrow 5' exonuclease and a poly(A) polymerase in Escherichia coli. Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11966–11971. doi: 10.1073/pnas.220295997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mullet J. E. Dynamic regulation of chloroplast transcription. Plant Physiol. 1993 Oct;103(2):309–313. doi: 10.1104/pp.103.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ow M. C., Liu Q., Kushner S. R. Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly. Mol Microbiol. 2000 Nov;38(4):854–866. doi: 10.1046/j.1365-2958.2000.02186.x. [DOI] [PubMed] [Google Scholar]
  35. Py B., Causton H., Mudd E. A., Higgins C. F. A protein complex mediating mRNA degradation in Escherichia coli. Mol Microbiol. 1994 Nov;14(4):717–729. doi: 10.1111/j.1365-2958.1994.tb01309.x. [DOI] [PubMed] [Google Scholar]
  36. Py B., Higgins C. F., Krisch H. M., Carpousis A. J. A DEAD-box RNA helicase in the Escherichia coli RNA degradosome. Nature. 1996 May 9;381(6578):169–172. doi: 10.1038/381169a0. [DOI] [PubMed] [Google Scholar]
  37. Régnier P., Arraiano C. M. Degradation of mRNA in bacteria: emergence of ubiquitous features. Bioessays. 2000 Mar;22(3):235–244. doi: 10.1002/(SICI)1521-1878(200003)22:3<235::AID-BIES5>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  38. Schuster G., Lisitsky I., Klaff P. Polyadenylation and degradation of mRNA in the chloroplast. Plant Physiol. 1999 Aug;120(4):937–944. doi: 10.1104/pp.120.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schägger H., von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991 Dec;199(2):223–231. doi: 10.1016/0003-2697(91)90094-a. [DOI] [PubMed] [Google Scholar]
  40. Shteiman-Kotler A., Schuster G. RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1. Nucleic Acids Res. 2000 Sep 1;28(17):3310–3315. doi: 10.1093/nar/28.17.3310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sohlberg B., Lundberg U., Hartl F. U., von Gabain A. Functional interaction of heat shock protein GroEL with an RNase E-like activity in Escherichia coli. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):277–281. doi: 10.1073/pnas.90.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Steege D. A. Emerging features of mRNA decay in bacteria. RNA. 2000 Aug;6(8):1079–1090. doi: 10.1017/s1355838200001023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stern D. B., Gruissem W. Control of plastid gene expression: 3' inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell. 1987 Dec 24;51(6):1145–1157. doi: 10.1016/0092-8674(87)90600-3. [DOI] [PubMed] [Google Scholar]
  44. Symmons M. F., Jones G. H., Luisi B. F. A duplicated fold is the structural basis for polynucleotide phosphorylase catalytic activity, processivity, and regulation. Structure. 2000 Nov 15;8(11):1215–1226. doi: 10.1016/s0969-2126(00)00521-9. [DOI] [PubMed] [Google Scholar]
  45. Tock M. R., Walsh A. P., Carroll G., McDowall K. J. The CafA protein required for the 5'-maturation of 16 S rRNA is a 5'-end-dependent ribonuclease that has context-dependent broad sequence specificity. J Biol Chem. 2000 Mar 24;275(12):8726–8732. doi: 10.1074/jbc.275.12.8726. [DOI] [PubMed] [Google Scholar]
  46. Vanzo N. F., Li Y. S., Py B., Blum E., Higgins C. F., Raynal L. C., Krisch H. M., Carpousis A. J. Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome. Genes Dev. 1998 Sep 1;12(17):2770–2781. doi: 10.1101/gad.12.17.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilm M., Shevchenko A., Houthaeve T., Breit S., Schweigerer L., Fotsis T., Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature. 1996 Feb 1;379(6564):466–469. doi: 10.1038/379466a0. [DOI] [PubMed] [Google Scholar]
  48. Yehudai-Resheff S., Hirsh M., Schuster G. Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts. Mol Cell Biol. 2001 Aug;21(16):5408–5416. doi: 10.1128/MCB.21.16.5408-5416.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES