Abstract
ALIX/Bro1 proteins are conserved in eukaryotes where they enable targeted trafficking of membrane-associated proteins through the late endosome route to the vacuole. For this, ALIX/Bro1 proteins associate with the endosomal sorting complex required for transport (ESCRT) machinery acting as ubiquitin receptors that recognize and sort protein cargoes by binding to ubiquitin–cargo conjugates. However, recent findings show direct interaction of ALIX and protein cargoes, pointing to the existence of different mechanisms for specific target recognition by ALIX. The catalogue of proteins that interact with the Arabidopsis homologue of ALIX is increasing, including both protein cargoes and regulatory proteins that mediate or modulate ALIX function. In this context, we describe a toolkit of techniques to analyze the effect of ALIX function in the endosomal trafficking of specific cargoes, which could be easily extended to other components of the plant ESCRT machinery.
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References
Odorizzi G (2006) The multiple personalities of Alix. J Cell Sci 119:3025–3032
Cardona-López X, Cuyas L, Marín E, Rajulu C, Irigoyen ML, Gil E, Puga MI, Bligny R, Nussaume L, Geldner N, Paz-Ares J, Rubio V (2015) ESCRT-III-associated protein ALIX mediates high-affinity phosphate transporter trafficking to maintain phosphate homeostasis in Arabidopsis. Plant Cell 27:2560–2581
Kalinowska K, Nagel MK, Goodman K, Cuyas L, Anzenberger F, Alkofer A, Paz-Ares J, Braun P, Rubio V, Otegui MS, Isono E (2015) Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3. Proc Natl Acad Sci U S A 112:E5543–E5551
García-León M, Cuyas L, Abd El-Moneim D, Rodriguez L, Belda-Palazon B, Sánchez-Quant E, Fernández Y, Roux B, Zamarreño AM, Garcia-Mina JM, Nussaume L, Rodriguez PL, Paz-Ares J, Leonhardt N, Rubio V (2019) Stomatal aperture and turnover of ABA receptors are regulated by Arabidopsis ALIX. Plant Cell 31(10). https://doi.org/10.1105/tpc.19.00399; pii: tpc.00399.2019
Mageswaran SK, Dixon MG, Curtiss M, Keener JP, Babst M (2014) Binding to any ESCRT can mediate ubiquitin-independent cargo sorting. Traffic 15:212–229
Belda-Palazon B, Rodriguez L, Fernandez MA, Castillo MC, Anderson EA, Gao C, González-Guzmán M, Peirats-Llobet M, Zhao Q, De Winne N, Gevaert K, De Jaeger G, Jiang L, Leon J, Mullen RT, Rodriguez PL (2016) FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates its Delivery to the Vacuolar Degradation Pathway. Plant Cell 28:2291–2311
Shen J, Gao C, Zhao Q, Lin Y, Wang X, Zhuang X, Jiang L (2016) AtBRO1 functions in ESCRT-I complex to regulate multivesicular body protein sorting. Mol Plant 9:760–763
Yu F, Lou L, Tian M, Li Q, Ding Y, Cao X, Wu Y, Belda-Palazon B, Rodriguez PL, Yang S, Xie Q (2016) ESCRT-I component VPS23A affects ABA signaling by recognizing ABA receptors for endosomal degradation. Mol Plant 9:1570–1582
Voinnet O (2003) RNA silencing bridging the gaps in wheat extracts. Trends Plant Sci 8:307–309
Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025
Azimzadeh J, Nacry P, Christodoulidou A, Drevensek S, Camilleri C, Amiour N, Parcy F, Pastuglia M, Bouchez D (2008) Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin. Plant Cell 20: 2146–2159
Acknowledgments
Research in our lab was supported by Grant BIO2016-80551-R funded by MINECO and AEI/FEDER/EU. M.G.-L. was supported by a FPI fellowship from the “Severo Ochoa” (BES2015-071820; Spanish Ministry of Education) and EMBO Short-Term (ASTF 7678-2016) programs. We are very thankful to the CNB-CSIC Confocal Microscopy facility for comments on the imaging parameters and procedures.
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García-León, M., Rubio, V. (2020). Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_5
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DOI: https://doi.org/10.1007/978-1-0716-0767-1_5
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