title: Using Photoconvertible and Extractable Fluorescent Proteins to Study Autophagy in Plants.
abstract: Several methodologies have been employed to understand the kinetics of induced autophagic degradation in plants, but most of them are not capable of distinguishing the autophagic cargo proteins before and after induction of autophagy in cells. Here, we designed a mass photoconverter that allowed us to simultaneously monitor protein synthesis and degradation in tobacco BY-2 cells using a photoconvertible fluorescence marker protein, Kikume Green Red (KikGR). An example of a new protocol for the analysis of autophagy progression using a fusion protein of cytochrome b5 and KikGR under phosphate starvation is described. The other example described is the analysis of the proliferation of Golgi apparatus in tobacco BY-2 cells using the fusion protein of a prolyl 4-hydroxylase NtP4H1.1 and monomeric KikGR. A detailed protocol on key analysis, as well as tips and notes for experiments using KikGR proteins, are described.
authors.affiliation: Laboratory of Plant Nutrition, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: M O
authors.initials: MO
authors.lastname: Abiodun
authors.affiliation: Laboratory of Plant Nutrition, Faculty of Agriculture, Kyushu University, Fukuoka, Japan; Biotron Application Center, Kyushu University, Fukuoka, Japan; Research Center for Organelle Homeostasis, Kyushu University, Fukuoka, Japan. Electronic address: kenmat@agr.kyushu-u.ac.jp.
authors.forename: K
authors.initials: K
authors.lastname: Matsuoka
mesh_terms: D001343:Autophagy; D002460:Cell Line; D015786:Cytochromes b5 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods; D055668:Photochemical Processes; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D064828:Prolyl Hydroxylases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D066329:Protein Aggregates; D014176:Protein Biosynthesis; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014170:Transformation, Genetic
chemical_list: D008164:Luminescent Proteins; D010940:Plant Proteins; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D064828:Prolyl Hydroxylases
keywords: Bulk photoconversion; Organelle turnover; Photoconvertible fluorescent protein; Protein aggregate; Quantitative analysis; Time-course experiment