title: A novel method for monitoring the localization of cytochromes P450 and other endoplasmic reticulum membrane associated proteins: a tool for investigating the formation of metabolons.
abstract: In plants and possibly other organisms, channelling of the reactive intermediates resulting from P450 oxygenation is thought to require the formation of supramolecular complexes associating membrane-bound and soluble enzymes. This implies a most probably loose membrane association of the soluble proteins. For the assessment of such membrane association in vivo, we propose an imaging strategy based on the accurate evaluation of fluorescent protein repartition and distance around endoplasmic reticulum (ER) tubules. It requires candidate protein fusion constructs with fluorescent reporters and transient expression in leaves of Nicotiana benthamiana. The method was tested with soluble eGFP/mRFP1, with various P450 and P450 reductase fluorescent fusions, and with anchored eGFP/mRFP1. It easily differentiated soluble and anchored proteins and detects subtle changes in ER tubules. The method was further assessed with a soluble protein previously shown to be loosely associated with the ER, the phenylalanine ammonia lyase PAL1 involved in the lignin biosynthetic pathway. This protein was found located in close vicinity to the ER. Taken together, these data indicate that the method proposed herein is suitable to monitor membrane association and relocalization of soluble proteins involved in the formation of metabolons.
authors.affiliation: Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, Strasbourg, France.
authors.forename: Jean-Etienne
authors.initials: JE
authors.lastname: Bassard
authors.forename: Jérôme
authors.initials: J
authors.lastname: Mutterer
authors.forename: Frédéric
authors.initials: F
authors.lastname: Duval
authors.forename: Danièle
authors.initials: D
authors.lastname: Werck-Reichhart
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism; D012680:Sensitivity and Specificity; D050564:Trans-Cinnamate 4-Monooxygenase / Q000378:metabolism
chemical_list: C511988:ATR1 protein, Arabidopsis; D029681:Arabidopsis Proteins; C493362:CYP73A5 protein, Arabidopsis; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D020598:Proto-Oncogene Proteins c-myb; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003577:Cytochrome P-450 Enzyme System; D050564:Trans-Cinnamate 4-Monooxygenase; D010650:Phenylalanine Ammonia-Lyase