Saccomanno Antonietta, Potocký Martin, Pejchar Přemysl, Hála Michal, Shikata Hiromasa, Schwechheimer Claus, Žárský Viktor
Laboratory of Cell Biology, Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czechia.
Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague, Czechia.
Front Plant Sci. 2021 Jan 14;11:609600. doi: 10.3389/fpls.2020.609600. eCollection 2020.
Exocyst is a heterooctameric protein complex crucial for the tethering of secretory vesicles to the plasma membrane during exocytosis. Compared to other eukaryotes, exocyst subunit EXO70 is represented by many isoforms in land plants whose cell biological and biological roles, as well as modes of regulation remain largely unknown. Here, we present data on the phospho-regulation of exocyst isoform EXO70C2, which we previously identified as a putative negative regulator of exocyst function in pollen tube growth. A comprehensive phosphoproteomic analysis revealed phosphorylation of EXO70C2 at multiple sites. We have now performed localization and functional studies of phospho-dead and phospho-mimetic variants of Arabidopsis EXO70C2 in transiently transformed tobacco pollen tubes and stably transformed Arabidopsis wild type and mutant plants. Our data reveal a dose-dependent effect of At overexpression on pollen tube growth rate and cellular architecture. We show that changes of the AtEXO70C2 phosphorylation status lead to distinct outcomes in wild type and mutant cells, suggesting a complex regulatory pattern. On the other side, phosphorylation does not affect the cytoplasmic localization of AtEXO70C2 or its interaction with putative secretion inhibitor ROH1 in the yeast two-hybrid system.
外泌体是一种异源八聚体蛋白复合物，在胞吐作用期间对分泌囊泡与质膜的拴系至关重要。与其他真核生物相比，陆地植物中，外泌体亚基EXO70由许多亚型代表，其细胞生物学和生物学作用以及调控模式在很大程度上仍不清楚。在此，我们展示了关于外泌体亚型EXO70C2磷酸化调控的数据，我们之前将其鉴定为花粉管生长中外泌体功能的假定负调控因子。全面的磷酸化蛋白质组学分析揭示了EXO70C2在多个位点的磷酸化。我们现在已经在瞬时转化的烟草花粉管以及稳定转化的拟南芥野生型和突变体植物中，对拟南芥EXO70C2的磷酸化缺失和磷酸化模拟变体进行了定位和功能研究。我们的数据揭示了AtEXO70C2过表达对花粉管生长速率和细胞结构的剂量依赖性影响。我们表明，AtEXO70C2磷酸化状态的变化在野生型和突变体细胞中导致不同的结果，表明存在复杂的调控模式。另一方面，磷酸化不影响AtEXO70C2在酵母双杂交系统中的细胞质定位或其与假定的分泌抑制剂ROH1的相互作用。
