Pharmaco-proteomic analysis of phosphatidylinositol 3/4-kinase inhibitor wortmannin in Arabidopsis
Diverse pharmaceutical drugs inhibiting vesicular transport were employed in previous genetic and cell biological studies. We recently reported that proteomic analysis of Arabidopsis roots treated by pharmaceutical inhibitor brefeldin A provides good potential to find novel cytoskeletal proteins regulating vesicular trafficking, such as the actin-binding protein profilin 2 (Takáč et al., 2011). In the current study we have used a similar approach to find regulatory proteins playing role in vacuolar sorting and endocytosis. For this aim we treated Arabidopsis roots with wortmannin, which is a fungal metabolite inhibiting phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinase (PI4K). This leads to the fusion, swelling and vacuolization of multivesicular bodies, and to the inhibition of endocytosis. Wortmannin-treated roots were subjected to comparative gel-based (2-D electrophoresis) and gel-free (2D LC MS/MS combined with label-free quantification) proteomic analyses.
Our results suggest that phospholipase D alpha 1 and CDC48 may regulate membrane fusion mechanism caused by wortmannin. The soluble vacuolar cargo proteins such as subtilase and cathepsin B-like cysteine protease were downregulated by wortmannin suggesting their possible degradation. Moreover, this proteomic analyses helped to identify small RabGTPase called RabA1d as a protein affected by wortmannin. Preliminary data from live cell imaging on GFP-tagged RabA1d revealed that fusion protein is likely released form endosomes to cytoplasm after wortmannin treatment. The tight interplay between cytoskeleton and vesicular trafficking is also documented in our study by downregulation of actin 8, while tubulin beta-4 chain was slightly upregulated by wortmannin. Additionally, actin binding phospholipase D alpha 1 was downregulated by wortmannin. We propose that this protein could serve as a linker of the PI3/4K–dependent vesicullar trafficking and cytoskeleton remodelling in Arabidopsis roots.
Takáč T, Pechan T, Richter H, Müller J, Eck C, Böhm N, Obert B, Ren H, Niehaus K, Šamaj J (2011) J.Proteome Res. 10: 488-501