Abstract
Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins.
Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.
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References
Rogers JC (1985) Two barley alpha-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260(6):3731–3738
Denecke J, Botterman J, Deblaere R (1990) Protein secretion in plant cells can occur via a default pathway. Plant Cell 2(1):51–59
Phillipson BA et al (2001) Secretory bulk flow of soluble proteins is efficient and COPII dependent. Plant Cell 13(9):2005–2020
Denecke J, De Rycke R, Botterman J (1992) Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope. EMBO J 11(6):2345–2355
Pimpl P et al (2006) Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway. Plant Cell 18(1):198–211
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
Holwerda BC, Padgett HS, Rogers JC (1992) Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions. Plant Cell 4(3):307–318
Frigerio L, de Virgilio M, Prada A, Faoro F, Vitale A (1998) Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide. Plant Cell 10(6):1031–1042
Koide Y, Hirano H, Matsuoka K, Nakamura K (1997) The N-terminal propeptide of the precursor to sporamin acts as a vacuole-targeting signal even at the C terminus of the mature part in tobacco cells. Plant Physiol 114(3):863–870
Pimpl P, Hanton SL, Taylor JP, Pinto-DaSilva LL, Denecke J (2003) The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole. Plant Cell 15(5):1242–1256
Bottanelli F, Foresti O, Hanton S, Denecke J (2011) Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo. Plant Cell 23(8):3007–3025
daSilva LL et al (2004) Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells. Plant Cell 16(7):1753–1771
daSilva LL et al (2005) Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. Plant Cell 17(1):132–148
Gershlick DC et al (2014) Golgi-dependent transport of vacuolar sorting receptors is regulated by COPII, AP1, and AP4 protein complexes in tobacco. Plant Cell 26(3):1308–1329
Langhans M et al (2008) In vivo trafficking and localization of p24 proteins in plant cells. Traffic 9(5):770–785
Langhans M, Niemes S, Pimpl P, Robinson DG (2009) Oryzalin bodies: in addition to its anti-microtubule properties, the dinitroaniline herbicide oryzalin causes nodulation of the endoplasmic reticulum. Protoplasma 236(1–4):73–84
Leborgne-Castel N, Jelitto-Van Dooren EP, Crofts AJ, Denecke J (1999) Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress. Plant Cell 11(3):459–470
Niemes S et al (2010) Sorting of plant vacuolar proteins is initiated in the ER. Plant J 62(4):601–614
Niemes S et al (2010) Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J 61(1):107–121
Pimpl P et al (2000) In situ localization and in vitro induction of plant COPI-coated vesicles. Plant Cell 12(11):2219–2236
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(6):1477–1497
Bubeck J et al (2008) The syntaxins SYP31 and SYP81 control ER-Golgi trafficking in the plant secretory pathway. Traffic 9(10):1629–1652
Shahriari M et al (2010) The AAA-type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana. Plant J 64(1):71–85
Künzl F, Früholz S, Fäßler F, Li B, Pimpl P (2016) Receptor-mediated sorting of soluble vacuolar proteins ends at the trans-Golgi network/early endosome. Nat Plants 2:16017
Humair D, Hernandez Felipe D, Neuhaus JM, Paris N (2001) Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor. Plant Cell 13(4):781–792
Scheuring D et al (2012) Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway. BMC Plant Biol 12:164
Acknowledgements
We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (PI 769/1-2 and the Collaborative Research Centre SFB 1101 “Molecular Encoding of Specificity in Plant Processes”) and of the German Academic Exchange Service (Project 57219822).
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Früholz, S., Pimpl, P. (2017). Analysis of Nanobody–Epitope Interactions in Living Cells via Quantitative Protein Transport Assays. In: Jiang, L. (eds) Plant Protein Secretion. Methods in Molecular Biology, vol 1662. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7262-3_15
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DOI: https://doi.org/10.1007/978-1-4939-7262-3_15
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