title: Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes.
abstract: The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.
authors.affiliation: Institute of Experimental Botany, Czech Academy of Sciences, Prague 6, Czech Republic (J.S., P.P., N.V., V.Ž., M.P.).|Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague 2, Czech Republic (J.S., V.Ž.); and.|Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 6, Czech Republic (J.Š.).
authors.forename: Juraj
authors.identifier: 0000-0002-4876-5454
authors.initials: J
authors.lastname: Sekereš
authors.affiliation: Institute of Experimental Botany, Czech Academy of Sciences, Prague 6, Czech Republic (J.S., P.P., N.V., V.Ž., M.P.).|Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague 2, Czech Republic (J.S., V.Ž.); and.|Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 6, Czech Republic (J.Š.).
authors.forename: Přemysl
authors.identifier: 0000-0003-0488-7465
authors.initials: P
authors.lastname: Pejchar
authors.affiliation: Institute of Experimental Botany, Czech Academy of Sciences, Prague 6, Czech Republic (J.S., P.P., N.V., V.Ž., M.P.).|Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague 2, Czech Republic (J.S., V.Ž.); and.|Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 6, Czech Republic (J.Š.).
authors.forename: Jiří
authors.initials: J
authors.lastname: Šantrůček
authors.affiliation: Institute of Experimental Botany, Czech Academy of Sciences, Prague 6, Czech Republic (J.S., P.P., N.V., V.Ž., M.P.).|Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague 2, Czech Republic (J.S., V.Ž.); and.|Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 6, Czech Republic (J.Š.).
authors.forename: Nemanja
authors.initials: N
authors.lastname: Vukašinović
authors.affiliation: Institute of Experimental Botany, Czech Academy of Sciences, Prague 6, Czech Republic (J.S., P.P., N.V., V.Ž., M.P.).|Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague 2, Czech Republic (J.S., V.Ž.); and.|Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 6, Czech Republic (J.Š.).
authors.forename: Viktor
authors.identifier: 0000-0002-5301-0339
authors.initials: V
authors.lastname: Žárský
authors.affiliation: Institute of Experimental Botany, Czech Academy of Sciences, Prague 6, Czech Republic (J.S., P.P., N.V., V.Ž., M.P.); potocky@ueb.cas.cz.|Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague 2, Czech Republic (J.S., V.Ž.); and potocky@ueb.cas.cz.|Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 6, Czech Republic (J.Š.) potocky@ueb.cas.cz.
authors.forename: Martin
authors.identifier: 0000-0002-3699-7549
authors.initials: M
authors.lastname: Potocký
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D018613:Microscopy, Confocal; D010802:Phylogeny; D010940:Plant Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D053205:Pollen Tube / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms