title: GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues.
abstract: The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.
authors.affiliation: Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.
authors.forename: Casper
authors.initials: C
authors.lastname: Søgaard
authors.forename: Anne
authors.initials: A
authors.lastname: Stenbæk
authors.forename: Sophie
authors.initials: S
authors.lastname: Bernard
authors.forename: Masood
authors.initials: M
authors.lastname: Hadi
authors.forename: Azeddine
authors.initials: A
authors.lastname: Driouich
authors.forename: Henrik Vibe
authors.initials: HV
authors.lastname: Scheller
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Sakuragi
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods*; D005936:Glucans / Q000096:biosynthesis; D006056:Golgi Apparatus / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009928:Organ Specificity; D018515:Plant Leaves / Q000166:cytology / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000096:biosynthesis*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D014026:Nicotiana / Q000166:cytology / Q000235:genetics; D014990:Xylans / Q000096:biosynthesis
chemical_list: D029681:Arabidopsis Proteins; D005936:Glucans; D008565:Membrane Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014990:Xylans; C030080:araban; C029353:xyloglucan