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

• Published in: The FEBS journal Vol. 279; no. 9; pp. 1576 - 1583
• Main Authors: Bassard, Jean‐Etienne, Mutterer, Jérôme, Duval, Frédéric, Werck‐Reichhart, Danièle
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.05.2012
• Subjects: Arabidopsis - enzymology; Arabidopsis Proteins - metabolism; Cytochrome P-450 Enzyme System - metabolism; endoplasmic reticulum; Endoplasmic Reticulum - enzymology; Fluorescent Dyes - metabolism; Green Fluorescent Proteins - metabolism; Macromolecular Substances - metabolism; membrane; Membrane Proteins - metabolism; metabolon; Microscopy, Confocal - methods; Phenylalanine Ammonia-Lyase - metabolism; protein localization; Proto-Oncogene Proteins c-myb - metabolism; Sensitivity and Specificity; Trans-Cinnamate 4-Monooxygenase - metabolism
• ISSN: 1742-464X; 1742-4658; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08312.x

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. Database accession numbers CYP51G1 (Uniprot Q9SAA9), PAL1 (Uniprot P25872), CYP98A3 (Uniprot O22203), CYP73A5 (Uniprot B1GV49), ATR1 (Uniprot Q9SB48), eGFP (GenBank DQ768212), mRFP1 (Uniprot Q9U6Y8) A novel tool for investigating the formation of metabolons from the information captured by confocal microscopy. The method is designed to evaluate protein distribution and distance around the ER tubules (ER‐FWHM). It is suitable for investigating the dynamics of protein‐membrane association, and for detection of loose association of soluble proteins.