Abstract
The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an α-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a β-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abedi MR, Camponigro G, Kamb A (1998) Green fluorescent protein as a scaffold for intracellular presentation of peptides. Nucleic Acids Res 26:623–630
Batoko H, Zheng HQ, Hawes C, Moore I (2000) A Rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants. Plant Cell 12(11):2201–2218
Brandizzi F, Frangne N, Marc-Martin S, Hawes C, Neuhaus JM, Paris N (2002) The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14(5):1077–1092
Bulbarelli A, Sprocati T, Barberi M, Pedrazzini E, Borgese N (2002) Trafficking of tail-anchored proteins: transport from the endoplasmic reticulum to the plasma membrane and sorting between surface domains in polarised epithelial cells. J Cell Sci 115:1689–1702
daSilva LL, Taylor JP, Hadlington JL, Hanton SL, Snowden CJ, Fox SJ, Foresti O, Brandizzi F, Denecke J (2005) Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. Plant Cell 17:132–148
daSilva LL, Foresti O, Denecke J (2006) Targeting of the plant vacuolar sorting receptor BP80 is dependent on multiple sorting signals in the cytosolic tail. Plant Cell 18:1477–1497
Di Sansebastiano GP, Paris N, Marc-Martin S, Neuhaus JM (1998) Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway. Plant J 15:449–457
Di Sansebastiano GP, Renna L, Piro G, Dalessandro G (2004) Stubborn GFPs in Nicotiana tabacum vacuoles. Plant Biosyst 138(1):37–42
Di Sansebastiano GP, Gigante M, De Domenico S, Piro G, Dalessandro G (2006) Sorting of GFP tagged NtSyr1, an ABA related syntaxin. Plant Signal Behav 1(2):76–84
Di Sansebastiano GP, Renna L, Gigante M, De Caroli M, Piro G, Dalessandro G (2007) Green fluorescent protein reveals variability in vacuoles of three plant species. Biol Plant 51(1):49–55
Dupree P, Sherrier DJ (1998) The Golgi apparatus. Biochim Biophys Acta 1404(1–2):259–270
Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Laine AC, Gomord V, Faye L (1998) N-glycoprotein biosynthesis: recent development and future trends. Plant Mol Biol 38(1–2):31–48
Leucci MR, Di Sansebastiano GP, Gigante M, Dalessandro G, Piro G (2007) Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways. Planta 225(4):1001–1017
Meder D, Shevchenko A, Simons K, Füllekrug J (2005) Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells. J Cell Biol 168(2):303–313
Petruccelli S, Otegui MS, Lareu F, Tran Dinh O, Fitchette AC, Circosta A, Rumbo M, Bardor M, Carcamo R, Gomord V, Beachy RN (2006) A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J 4(5):511–527
Rehman RU, Stigliano E, Lycett GW, Sticher L, Sbano F, Faraco M, Dalessandro G, Di Sansebastiano GP (2008) Tomato Rab11a characterization evidenced a difference between SYP121-dependent and SYP122-dependent exocytosis. Plant Cell Physiol 49(5):751–766
Saint-Jore-Dupas C, Nebenführ A, Boulaflous A, Follet-Gueye ML, Plasson C, Hawes C, Driouich A, Faye L, Gomord V (2006) Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway. Plant Cell 18(11):3182–3200
Saint-Jore-Dupas C, Faye L, Gomord V (2007) From planta to pharma with glycosylation in the toolbox. Trends Biotechnol 25(7):317–323
Schwarzerová K, Petrásek J, Panigrahi KC, Zelenková S, Opatrný Z, Nick P (2006) Intranuclear accumulation of plant tubulin in response to low temperature. Protoplasma 227(2–4):185–196
Sohn E, Rojas-Pierce M, Pan S, Carter C, Serrano-Mislata A, Madueno F, Rojo E, Surpin M, Raikhel NV (2007) The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole. Proc Natl Acad Sci USA 104:18801–18806
Staehelin LA, Moore I (1995) The plant Golgi apparatus: structure, functional organization and trafficking mechanisms. Annu Rev Plant Physiol Plant Mol Biol 46:261–288
Staudacher E, Altmann F, Wilson IBH, März L (1999) Fucose in N-glycans: from plant to man. Biochim Biophys Acta 1473(1):216–236
Verweij W, Di Sansebastiano GP, Quattrocchio F, Dalessandro G (2008) Agrobacterium-mediated transient expression of vacuolar GFPs in Petunia leaves and petals. Plant Biosyst 142(2):343–347
Wilkins TA, Bednarek SY, Raikhel NV (1990) Role of propeptide glycan in posttranslational processing and transport of barley lectin to vacuoles in transgenic tobacco. Plant Cell 2:301–313
Acknowledgments
We thank the Italian Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST-PRIN2007) for funding to GPDS and GD and the European FP6 (MTKD-CT-2004-509253) for funding to KW. This work was also supported by the Swiss National Science Foundation grants 31-46926.96 for funding to JMN.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by F. Brandizzi.
Rights and permissions
About this article
Cite this article
Paris, N., Saint-Jean, B., Faraco, M. et al. Expression of a glycosylated GFP as a bivalent reporter in exocytosis. Plant Cell Rep 29, 79–86 (2010). https://doi.org/10.1007/s00299-009-0799-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-009-0799-7