Protein Targeting to the Plastid of Euglena

  • Dion G. Durnford
  • Steven D. SchwartzbachEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 979)


The lateral transfer of photosynthesis between kingdoms through endosymbiosis is among the most spectacular examples of evolutionary innovation. Euglena, which acquired a chloroplast indirectly through an endosymbiosis with a green alga, represents such an example. As with other endosymbiont-derived plastids from eukaryotes, there are additional membranes that surround the organelle, of which Euglena has three. Thus, photosynthetic genes that were transferred from the endosymbiont to the host nucleus and whose proteins are required in the new plastid, are now faced with targeting and plastid import challenges. Early immunoelectron microscopy data suggested that the light-harvesting complexes, photosynthetic proteins in the thylakoid membrane, are post-translationally targeted to the plastid via the Golgi apparatus, an unexpected discovery at the time. Proteins targeted to the Euglena plastid have complex, bipartite presequences that direct them into the endomembrane system, through the Golgi apparatus and ultimately on to the plastid, presumably via transport vesicles. From transcriptome sequencing, dozens of plastid-targeted proteins were identified, leading to the identification of two different presequence structures. Both have an amino terminal signal peptide followed by a transit peptide for plastid import, but only one of the two classes of presequences has a third domain—the stop transfer sequence. This discovery implied two different transport mechanisms; one where the protein was fully inserted into the lumen of the ER and another where the protein remains attached to, but effectively outside, the endomembrane system. In this review, we will discuss the biochemical and bioinformatic evidence for plastid targeting, discuss the evolution of the targeting system, and ultimately provide a working model for the targeting and import of proteins into the plastid of Euglena.


Chloroplast protein import Complex plastids Endosymbiosis Euglena Plastid evolution Precursor protein Signal peptide Transit peptide 



Chloroplast endoplasmic reticulum


Carboxy terminal


Endoglycosidase H


Endoplasmic reticulum


ER associated degradation


Expressed sequence tag


Light-harvesting complex


Light harvesting chlorophyll a/b binding protein of photosystem II


Asparagine linked


N-ethylmaleimide-Sensitive factor


Amino terminal


Soluble peridinin-chlorophyll a-protein


Precursor to the light harvesting chlorophyll a/b binding protein of photosystem II


Precursor to the 30-kDa subunit of the oxygen-evolving complex


Precursor to the small subunit of ribulose bisphosphate carboxylase-oxygenase


Ribulose bisphosphate carboxylase-oxygenase


Small subunit of ribulose bisphosphate carboxylase-oxygenase


Symbiont-derived ERAD-like machinery


Soluble NSF attachment protein receptor


Translocon at the inner chloroplast envelope


Translocon at the outer chloroplast envelope


  1. Agrawal S, van Dooren GG, Beatty WL, Striepen B (2009) Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins. J Biol Chem 284(48):33683–33691PubMedPubMedCentralCrossRefGoogle Scholar
  2. Archibald JM (2015) Genomic perspectives on the birth and spread of plastids. Proc Natl Acad Sci U S A 112(33):10147–10153PubMedPubMedCentralCrossRefGoogle Scholar
  3. Bassham DC, Bartling D, Mould RM, Dunbar B, Weisbeek P, Herrmann RG, Robinson C (1991) Transport of proteins into chloroplasts. Delineation of envelope “transit” and thylakoid “transfer” signals within the pre-sequences of three imported thylakoid lumen proteins. J Biol Chem 266(35):23606–23610PubMedGoogle Scholar
  4. Bhaya D, Grossman AR (1991) Targeting proteins to diatom plastids involves transport through an endoplasmic reticulum. Mol Gen Genet 229:400–404PubMedCrossRefGoogle Scholar
  5. Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and fusion. Cell 116(2):153–166PubMedCrossRefGoogle Scholar
  6. Brandt P, von Kessel B (1983) Cooperation of cytoplasmic and plastidial translation in formation of the photosynthetic apparatus and its stage-specific efficiency. Plant Physiol 72(3):616–619PubMedPubMedCentralCrossRefGoogle Scholar
  7. Braulke T, Bonifacino JS (2009) Sorting of lysosomal proteins. Biochim Biophys Acta 1793(4):605–614PubMedCrossRefGoogle Scholar
  8. Bruce BD (2000) Chloroplast transit peptides: structure, function and evolution. Trends Cell Biol 10(10):440–447PubMedCrossRefGoogle Scholar
  9. Buren S, Ortega-Villasante C, Blanco-Rivero A, Martinez-Bernardini A, Shutova T, Shevela D, Messinger J, Bako L, Villarejo A, Samuelsson G (2011) Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana. PLoS One 6(6)Google Scholar
  10. Burki F (2014) The eukaryotic tree of life from a global phylogenomic perspective. Cold Spring Harb Perspect Biol 6(5):a016147PubMedPubMedCentralCrossRefGoogle Scholar
  11. Cavalier-Smith T (2003) Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae). Philos Trans R Soc Lond Ser B Biol Sci 358(1429):109–133CrossRefGoogle Scholar
  12. Chan RL, Keller M, Canaday J, Weil JH, Imbault P (1990) Eight small subunits of Euglena ribulose 1-5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein. EMBO J 9(2):333–338PubMedPubMedCentralGoogle Scholar
  13. Chauhan JS, Rao A, Raghava GP (2013) In silico platform for prediction of N-, O- and C-glycosites in eukaryotic protein sequences. PLoS One 8(6):e67008PubMedPubMedCentralCrossRefGoogle Scholar
  14. Durnford DG, Gray MW (2006) Analysis of Euglena gracilis plastid-targeted proteins reveals different classes of transit sequences. Eukaryot Cell 5(12):2079–2091PubMedPubMedCentralCrossRefGoogle Scholar
  15. Enomoto T, Sulli C, Schwartzbach SD (1997) A soluble chloroplast protease processes the Euglena polyprotein precursor to the light harvesting chlorophyll a/b binding protein of photosystem II. Plant Cell Physiol 38:743–746CrossRefGoogle Scholar
  16. Felsner G, Sommer MS, Gruenheit N, Hempel F, Moog D, Zauner S, Martin W, Maier UG (2011) ERAD components in organisms with complex red plastids suggest recruitment of a preexisting protein transport pathway for the periplastid membrane. Genome Biol Evol 3:140–150PubMedCrossRefGoogle Scholar
  17. Gibbs SP (1970) Comparative ultrastructure of algal chloroplast. Ann N Y Acad Sci 175(2):454–473CrossRefGoogle Scholar
  18. Gibbs SP (1978) The chloroplasts of Euglena may have evolved from symbiotic green algae. Can J Bot 56:2883–2889CrossRefGoogle Scholar
  19. Gibbs SP (1981a) The chloroplast endoplasmic reticulum: structure, function and evolutionary significance. Int Rev Cytol 72:49–99CrossRefGoogle Scholar
  20. Gibbs SP (1981b) The chloroplasts of some algal groups may have evolved from endosymbiotic eukaryotic algae. Ann N Y Acad Sci 361:193–207PubMedCrossRefGoogle Scholar
  21. Gray MW, Doolittle WF (1982) Has the endosymbiont hypothesis been proven? Microbiol Rev 46(1):1–42PubMedPubMedCentralGoogle Scholar
  22. Grossman A, Manodori A, Snyder D (1990) Light-harvesting proteins of diatoms: their relationship to the chlorophyll a/b binding proteins of higher plants and their mode of transport into plastids. Mol Gen Genet 224(1):91–100PubMedCrossRefGoogle Scholar
  23. von Heijne G (1988) Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta 947:307–333CrossRefGoogle Scholar
  24. Henze K, Badr A, Wettern M, Cerff R, Martin W (1995) A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution. Proc Natl Acad Sci U S A 92(20):9122–9126PubMedPubMedCentralCrossRefGoogle Scholar
  25. Houlne G, Schantz R (1987) Molecular analysis of the transcripts encoding the light-harvesting chlorophyll a/b protein in Euglena gracilis: unusual size of the mRNA. Curr Genet 12(8):611–616PubMedCrossRefGoogle Scholar
  26. Houlne G, Schantz R (1988) Characterization of cDNA sequences for LHCI apoproteins in Euglena gracilis: the mRNA encodes a large precursor containing several consecutive divergent polypeptides. Mol Gen Genet 213(2–3):479–486PubMedCrossRefGoogle Scholar
  27. Inagaki J, Fujita Y, Hase T, Yamamoto Y (2000) Protein translocation within chloroplast is similar in Euglena and higher plants. Biochem Biophys Res Commun 277(2):436–442PubMedCrossRefGoogle Scholar
  28. Keeling PJ (2013) The number, speed, and impact of plastid endosymbioses in eukaryotic evolution. Annu Rev Plant Biol 64:583–607PubMedCrossRefGoogle Scholar
  29. Keller M, Chan RL, Tessier LH, Weil JH, Imbault P (1991) Post-transcriptional regulation by light of the biosynthesis of Euglena ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit. Plant Mol Biol 17(1):73–82PubMedCrossRefGoogle Scholar
  30. Kishore R, Schwartzbach SD (1992) Translational control of the synthesis of the Euglena light harvesting chlorophyll a/b binding protein of photosystem II. Plant Sci 85:79–89CrossRefGoogle Scholar
  31. Kishore R, Muchhal U, Schwartzbach SD (1993) The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum targeting domain. Proc Natl Acad Sci U S A 90:11845–11849PubMedPubMedCentralCrossRefGoogle Scholar
  32. Ko K, Cashmore AR (1989) Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein. EMBO J 8(11):3187–3194PubMedPubMedCentralGoogle Scholar
  33. Koziol AG, Durnford DG (2008) Euglena light-harvesting complexes are encoded by multifarious polyprotein mRNAs that evolve in concert. Mol Biol Evol 25(1):92–100PubMedCrossRefGoogle Scholar
  34. Kuroiwa T, Sakaguchi M, Mihara K, Omura T (1991) Systematic analysis of stop-transfer sequence for microsomal membrane. J Biol Chem 266(14):9251–9255PubMedGoogle Scholar
  35. Lee J, Kim DH, Hwang I (2014) Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria. Front Plant Sci 5:173PubMedPubMedCentralGoogle Scholar
  36. Lefortran M, Pineau B (1980) Structure and functional-organization of chloroplastic membranes envelope in Euglena-gracilis. Eur J Cell Biol 22(1):279–279Google Scholar
  37. Lefort-Tran M, Pouphile M, Freyssinet G, Pineau B (1980) Structural and functional significance of the chloroplast envelope of Euglena: immunocytological and freeze fracture study. J Ultrastruct Res 73(1):44–63PubMedCrossRefGoogle Scholar
  38. Lin Q, Ma L, Burkhart W, Spremulli LL (1994) Isolation and characterization of cDNA clones for chloroplast translational initiation factor-3 from Euglena gracilis. J Biol Chem 269(13):9436–9444PubMedGoogle Scholar
  39. Lousa CD, Denecke J (2016) Lysosomal and vacuolar sorting: not so different after all! Biochem Soc Trans 44:891–897CrossRefGoogle Scholar
  40. Maier UG, Zauner S, Hempel F (2015) Protein import into complex plastids: cellular organization of higher complexity. Eur J Cell Biol 94(7–9):340–348PubMedCrossRefGoogle Scholar
  41. Muchhal US, Schwartzbach SD (1992) Characterization of a Euglena gene encoding a polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II. Plant Mol Biol 18(2):287–299PubMedCrossRefGoogle Scholar
  42. Muchhal US, Schwartzbach SD (1994) Characterization of the unique intron-exon junctions of Euglena gene(s) encoding the polyprotein precursor to the light-harvesting chlorophyll a/b binding protein of photosystem II. Nucl Acid Res 22:5737–5744CrossRefGoogle Scholar
  43. Nassoury N, Cappadocia M, Morse D (2003) Plastid ultrastructure defines the protein import pathway in dinoflagellates. J Cell Sci 116(Pt 14):2867–2874PubMedCrossRefGoogle Scholar
  44. Needham PG, Brodsky JL (2013) How early studies on secreted and membrane protein quality control gave rise to the ER associated degradation (ERAD) pathway: the early history of ERAD. Biochim Biophys Acta 1833(11):2447–2457PubMedPubMedCentralCrossRefGoogle Scholar
  45. Neuhaus JM, Rogers JC (1998) Sorting of proteins to vacuoles in plant cells. Plant Mol Biol 38:127–144PubMedCrossRefGoogle Scholar
  46. Osafune T, Schiff JA, Hase E (1990) Immunogold localization of LHCPII apoprotein in the Golgi of Euglena. Cell Struct Funct 15(2):99–105CrossRefGoogle Scholar
  47. Osafune T, Schiff JA, Hase E (1991a) Stage-dependent localization of LHCP II apoprotein in the Golgi of synchronized cells of Euglena gracilis by immunogold electron microscopy. Exp Cell Res 193(2):320–330PubMedCrossRefGoogle Scholar
  48. Osafune T, Sumida S, Schiff JA, Hase E (1991b) Immunolocalization of LHCPII apoprotein in the Golgi during light-induced chloroplast development in non-dividing Euglena cells. J Electron Microsc 40:41–47Google Scholar
  49. Paila YD, Richardson LGL, Schnell DJ (2015) New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development. J Mol Biol 427(5):1038–1060PubMedCrossRefGoogle Scholar
  50. Patron NJ, Waller RF (2007) Transit peptide diversity and divergence: a global analysis of plastid targeting signals. BioEssays 29(10):1048–1058PubMedCrossRefGoogle Scholar
  51. Patron NJ, Waller RF, Archibald JM, Keeling PJ (2005) Complex protein targeting to dinoflagellate plastids. J Mol Biol 348(4):1015–1024PubMedCrossRefGoogle Scholar
  52. Peschke M, Moog D, Klingl A, Maier UG, Hempel F (2013) Evidence for glycoprotein transport into complex plastids. Proc Natl Acad Sci U S A 110(26):10860–10865PubMedPubMedCentralCrossRefGoogle Scholar
  53. Plaumann M, Pelzer-Reith B, Martin WF, Schnarrenberger C (1997) Multiple recruitment of class-I aldolase to chloroplasts and eubacterial origin of eukaryotic class-II aldolases revealed by cDNAs from Euglena gracilis. Curr Genet 31(5):430–438PubMedCrossRefGoogle Scholar
  54. Rikin A, Schwartzbach SD (1988) Extremely large and slowly processed precursors to the Euglena light harvesting chlorophyll a/b binding proteins of photosystem II. Proc Natl Acad Sci U S A 85:5117–5121PubMedPubMedCentralCrossRefGoogle Scholar
  55. Rikin A, Schwartzbach SD (1989) Regulation by light and ethanol of the synthesis of the light harvesting chlorophyll a/b binding protein of photosystem II in Euglena. Planta 178:76–83PubMedCrossRefGoogle Scholar
  56. Santillan Torres JL, Atteia A, Claros MG, Gonzalez-Halphen D (2003) Cytochrome f and subunit IV, two essential components of the photosynthetic bf complex typically encoded in the chloroplast genome, are nucleus-encoded in Euglena gracilis. Biochim Biophys Acta 1604(3):180–189PubMedCrossRefGoogle Scholar
  57. Schiff JA, Schwartzbach SD, Osafune T, Hase E (1991) Photocontrol and processing of LHCPII apoprotein in Euglena - possible role of Golgi and other cytoplasmic sites. J Photochem Photobiol B Biol 11(2):219–236CrossRefGoogle Scholar
  58. Schwartzbach SD, Osafune T, Löffelhardt W (1998) Protein import into Cyanelles and complex chloroplasts. Plant Mol Biol 38:247–263PubMedCrossRefGoogle Scholar
  59. Shao S, Hegde RS (2011) Membrane protein insertion at the endoplasmic reticulum. Annu Rev Cell Dev Biol 27:25–56PubMedPubMedCentralCrossRefGoogle Scholar
  60. Sharif AL, Smith AG, Abell C (1989) Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena. Eur J Biochem 184(2):353–359PubMedCrossRefGoogle Scholar
  61. Shigemori Y, Inagaki J, Mori H, Nishimura M, Takahashi S, Yamamoto Y (1994) The presequence of the precursor to the nucleus-encoded 30 kDa protein of photosystem II in Euglena gracilis Z includes two hydrophobic domains. Plant Mol Biol 24(1):209–215PubMedCrossRefGoogle Scholar
  62. Slavikova S, Vacula R, Fang Z, Ehara T, Osafune T, Schwartzbach SD (2005) Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena. J Cell Sci 118(Pt 8):1651–1661PubMedCrossRefGoogle Scholar
  63. Smeekens S, Bauerle C, Hageman J, Keegstra K, Weisbeek P (1986) The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell 46(3):365–375PubMedCrossRefGoogle Scholar
  64. Spano AJ, Ghaus H, Schiff JA (1987) Chlorophyll-protein complexes and other thylakoid components at the low intensity threshold in Euglena chloroplast development. Plant Cell Physiol 28(6):1101–1108Google Scholar
  65. Stork S, Moog D, Przyborski JM, Wilhelmi I, Zauner S, Maier UG (2012) Distribution of the SELMA translocon in secondary plastids of red algal origin and predicted uncoupling of ubiquitin-dependent translocation from degradation. Eukaryot Cell 11(12):1472–1481PubMedPubMedCentralCrossRefGoogle Scholar
  66. Sulli C, Schwartzbach SD (1995) The polyprotein precursor to the Euglena light harvesting chlorophyll a/b-binding protein is transported to the Golgi apparatus prior to chloroplast import and polyprotein processing. J Biol Chem 270:13084–13090PubMedCrossRefGoogle Scholar
  67. Sulli C, Schwartzbach SD (1996) A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor. Plant Cell 8:43–53PubMedPubMedCentralCrossRefGoogle Scholar
  68. Sulli C, Fang ZW, Muchhal U, Schwartzbach SD (1999) Topology of Euglena chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast transport vesicles. J Biol Chem 274:457–463PubMedCrossRefGoogle Scholar
  69. Theg SM, Bauerle C, Olsen LJ, Selman BR, Keegstra K (1989) Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J Biol Chem 264(12):6730–6736PubMedGoogle Scholar
  70. Turmel M, Gagnon MC, O’Kelly CJ, Otis C, Lemieux C (2009) The chloroplast genomes of the green algae Pyramimonas, Monomastix, and Pycnococcus shed new light on the evolutionary history of prasinophytes and the origin of the secondary chloroplasts of euglenids. Mol Biol Evol 26(3):631–648PubMedCrossRefGoogle Scholar
  71. Vacula R, Steiner JM, Krajcovic J, Ebringer L, Loffelhardt W (1999) Nucleus-encoded precursors to thylakoid lumen proteins of Euglena gracilis possess tripartite presequences. DNA Res 6(1):45–49PubMedCrossRefGoogle Scholar
  72. Villarejo A, Buren S, Larsson S, Dejardin A, Monne M, Rudhe C, Karlsson J, Jansson S, Lerouge P, Rolland N, von Heijne G, Grebe M, Bako L, Samuelsson G (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat Cell Biol 7(12):1224–1231PubMedCrossRefGoogle Scholar
  73. Whatley JM, Whatley FR (1981) Chloroplast evolution. New Phytol 87(2):233–247CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Department of BiologyUniversity of New BrunswickFrederictonCanada
  2. 2.Department of Biological SciencesUniversity of MemphisMemphisUSA

Personalised recommendations