Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.
Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.
CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic. Electronic address: tomasz.nodzynski@ceitec.muni.cz.
Tomasz
T
Nodzyński
Department of Plant Systems Biology, VIB, 9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium.
Steffen
S
Vanneste
CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.
Marta
M
Zwiewka
CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic; National Centre for Biomolecular Research, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.
Markéta
M
Pernisová
CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic; National Centre for Biomolecular Research, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.
Jan
J
Hejátko
Institute of Science and Technology Austria (IST Austria), Am Campus 1, 3400 Klosterneuburg, Austria. Electronic address: jiri.friml@ist.ac.at.
Jiří
J
Friml
D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007210:Indoleacetic Acids / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D021381:Protein Transport
D029681:Arabidopsis Proteins; D007210:Indoleacetic Acids; D026901:Membrane Transport Proteins; C116363:PIN1 protein, Arabidopsis
Arabidopsis thaliana; auxin efflux carriers; plasma membrane protein; topology