Prabhakaran Mariyamma Neema, Hou Hongwei, Carland Francine M, Nelson Timothy, Schultz Elizabeth A
Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, TIK 3M4, Canada.
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
J Exp Bot. 2017 Jun 15;68(13):3375-3390. doi: 10.1093/jxb/erx180.
When FORKED1 (FKD1) is mutated, asymmetric localization of PINFORMED1 (PIN1), particularly to the apical side of cells, fails to occur properly in developing veins, resulting in an open vein pattern. FKD1 encodes a protein with a Pleckstrin homology-like (PL) domain, suggesting interaction with phosphoinositides. FKD1 has been previously found to interact with an ADP ribosylation factor GTPase-activating protein (ARF-GAP) important for vein patterning, SCARFACE/VAN3 (SFC). We find that FKD1-green fluorescent protein (GFP) localizes to the plasma membrane and to punctae labeled by SFC-yellow fluorescent protein (YFP). Supporting the idea that the FKD1 PL domain recognizes phosphatidylinositol 4-phosphate [PtdIns(4)P], FKD1-GFP co-localizes with PtdIns(4)P markers, and is more cytosolic when in a background mutant for the PtdIns(4,5)P2 hydrolases CVP2 and CVL1. Both FKD1 and SFC partially co-localize with markers for the trans-Golgi network (TGN), at which endocytic and secretory pathways merge. FKD1-labeled punctae rarely co-localize with the endocytic marker FM4-64, suggesting that FKD1 is not involved primarily in the endocytic pathway. FKD1 and SFC co-localize with members of the RABA group of RAB-GTPases, which are proposed to act in the post-Golgi secretory pathway. The compartments labeled by FKD1 and SFC do not localize to membrane compartments induced by the fungal toxin brefeldin A (BFA). Collectively, our data suggest that FKD1 and SFC act in a BFA-insensitive secretory pathway.
当FORKED1（FKD1）发生突变时，在发育中的叶脉中，PINFORMED1（PIN1）的不对称定位，尤其是向细胞顶端侧的定位，无法正常发生，导致叶脉模式开放。FKD1编码一种具有类普列克底物蛋白同源（PL）结构域的蛋白质，提示其与磷酸肌醇相互作用。先前已发现FKD1与对叶脉模式形成很重要的一种ADP核糖基化因子GTP酶激活蛋白（ARF-GAP）——SCARFACE/VAN3（SFC）相互作用。我们发现FKD1-绿色荧光蛋白（GFP）定位于质膜以及由SFC-黄色荧光蛋白（YFP）标记的点状结构。支持FKD1 PL结构域识别磷脂酰肌醇4-磷酸[PtdIns(4)P]这一观点的是，FKD1-GFP与PtdIns(4)P标记物共定位，并且在磷脂酰肌醇4,5-二磷酸[PtdIns(4,5)P2]水解酶CVP2和CVL1的背景突变体中更多地位于胞质中。FKD1和SFC都与反式高尔基体网络（TGN）的标记物部分共定位，内吞和分泌途径在TGN处交汇。FKD1标记的点状结构很少与内吞标记物FM4-64共定位，这表明FKD1主要不参与内吞途径。FKD1和SFC与RAB-GTPases的RABA亚组成员共定位，这些成员被认为在后高尔基体分泌途径中起作用。由FKD1和SFC标记的区室并不定位于由真菌毒素布雷菲德菌素A（BFA）诱导的膜区室。总体而言，我们的数据表明FKD1和SFC在对BFA不敏感的分泌途径中起作用。
