Abstract
ESCRT (Endosomal Sorting Complex Required for Transport) proteins are required for the sorting of biosynthetic and endocytic proteins at multivesicular bodies (MVBs). Here, I describe an assay to evaluate conservation of endosomal sorting functions of plant ESCRT proteins by trans-species complementation analysis. The assay is based on the imaging of a fluorescent biosynthetic MVB cargo, the carboxypeptidase S (CPS) fused to the green fluorescent protein GFP, in yeast ESCRT mutants expressing putative plant orthologues of the missing yeast ESCRT components.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH (1992) Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 3:1389–1402
Katsumata T, Hasegawa A, Fujiwara T, Komatsu T, Notomi M, Abe H, Natsume M, Kawaide H (2008) Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone brassinosteroids. Biosci Biotechnol Biochem 72:2110–2117
Babst M, Katzmann DJ, Estepa-Sabal EJ, Meerloo T, Emr SD (2002) ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting. Dev Cell 3:271–282
Babst M, Katzmann DJ, Snyder WB, Wendland B, Emr SD (2002) Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev Cell 3:283–289
Bilodeau PS, Winistorfer SC, Kearney WR, Robertson AD, Piper RC (2003) Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome. J Cell Biol 163:237–243
Katzmann DJ, Stefan CJ, Babst M, Emr SD (2003) Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J Cell Biol 162:413–423
Bankaitis VA, Johnson LM, Emr SD (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A 83:9075–9079
Rothman JH, Stevens TH (1986) Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell 47:1041–1051
Azmi I, Davies B, Dimaano C, Payne J, Eckert D, Babst M, Katzmann DJ (2006) Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J Cell Biol 172:705–717
Haas TJ, Sliwinski MK, Martínez DE, Preuss M, Ebine K, Ueda T, Nielsen E, Odorizzi G, Otegui MS (2007) The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19:1295–1312
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Reyes, F.C. (2014). Trans-species Complementation Analysis to Study Function Conservation of Plant Endosomal Sorting Complex Required for Transport (ESCRT) Proteins. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 1209. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1420-3_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1420-3_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1419-7
Online ISBN: 978-1-4939-1420-3
eBook Packages: Springer Protocols