title: The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes.
abstract: The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM-OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Hardré
authors.affiliation: Laboratoire de Biologie à Grande Echelle, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Kuhn
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Albrieux
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Juliette
authors.initials: J
authors.lastname: Jouhet
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Morgane
authors.initials: M
authors.lastname: Michaud
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Daphné
authors.initials: D
authors.lastname: Seigneurin-Berny
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Falconet
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Maryse A
authors.initials: MA
authors.lastname: Block
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, iRTSV CEA Grenoble, Grenoble, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Maréchal
keywords: biotinylation; chloroplast envelope; membrane protein complexes; outer envelope protein; thermolysin