Sorting inhibitors (Sortins): Chemical compounds to study vacuolar sorting in Arabidopsis

Abstract

Chemical genomics is an interdisciplinary approach that unites the power of chemical screens and genomics strategies to dissect biological processes such as endomembrane trafficking. We have taken advantage of the evolutionary conservation between plants and Saccharomyces cerevisiae to identify such chemicals. Using S. cerevisiae, we screened a library of diverse chemical structures for compounds that induce the secretion of carboxypeptidase Y, which is normally targeted to the vacuole. Among 4,800 chemicals screened, 14 compounds, termed sorting inhibitors (Sortins), were identified that stimulated secretion in yeast. In Arabidopsis seedlings, application of Sortin1 and -2 led to reversible defects in vacuole biogenesis and root development. Sortin1 was found to redirect the vacuolar destination of plant carboxypeptidase Y and other proteins in Arabidopsis suspension cells and cause these proteins to be secreted. Sortin1 treatment of whole Arabidopsis seedlings also resulted in carboxypeptidase Y secretion, indicating that the drug has a similar mode of action in cells and intact plants. We have demonstrated that screening of a simple eukaryote, in which vacuolar biogenesis is not essential, can be a powerful tool to find chemicals that interfere with vacuolar delivery of proteins in plants, where vacuole biogenesis is essential. Our studies were done by using a sublethal dose of Sortin1, demonstrating the powerful ability of the chemical to control the induced phenotype in a manner that would be difficult to achieve using conventional genetics.

Fig. 1.

Sortins trigger CPY secretion in yeast. Shown are chemical structures of Sortin1 (A, MW 441.44), Sortin2 (B, MW 429.92), and Sortin3 (C, MW 391.32). A dot-blot assay indicated that Sortin1 (D) and Sortin2 (E) generated strong vps phenotypes. In addition to a weak vps phenotype (F), Sortin3 induced a severe vam phenotype (H), similar to that of Class C vps yeast mutant vps16Δ (I). The tonoplast morphology of untreated yeast is shown (G). Concentrations are as indicated. (Scale bars = 5 μm.)

Fig. 2.

Sortin1 and Sortin2 alter vacuolar morphology in Arabidopsis seedlings. Seedlings expressing EGFP:δ-TIP as a tonoplast marker were germinated in the absence of Sortins (A) or in the presence of 227 μM Sortin1 (B)or233 μM Sortin2 (C). Vacuolar morphology in hypocotyls of 1-week-old seedlings was examined by confocal microscopy. Plants were then transferred onto nondrug medium to demonstrate reversibility of the phenotype induced by Sortin1 (D) and Sortin2 (E). Images in D and E (left to right) were taken at 0, 1, 2, 3, 4, and 5 days after transfer to non-drug medium. (Scale bars = 20 μmin A–C and 3 mm in D and E.)

Fig. 3.

Sortin1 induces secretion of the AtCPY precursor in Arabidopsis cell suspensions. The viability of Arabidopsis (A–C) and tobacco BY2 (D–F) suspension cells that were untreated (A and D) or treated with 57 μM Sortin1 (B and E)or58 μM Sortin2 (C and F) was analyzed by using fluorescein-diacetate. (G) Proteins secreted into the growth media of Arabidopsis suspension cells that were untreated (lane 1) or treated with Sortin1 (lane 2) were concentrated and analyzed by SDS/PAGE. Growth medium samples were also analyzed by Western blot (control, lane 5; Sortin1, lane 6). The AtCPY processing pattern was examined in cell pellets (control, lane 3; Sortin1, lane 4). The positions of the 60-kDa precursor (p), 48-kDa intermediate (i), and 24-kDa mature (m) forms of AtCPY are indicated. (H) A pulse–chase experiment tracking the AtCPY precursor secretion in cells that were untreated (control) or treated during the chase period with Sortin1 (Sortin1). (Scale bar = 500 μmin A–F.)

Fig. 4.

AtCPY is secreted into the apoplast in Sortin1-treated 1-week-old Arabidopsis seedlings. Immunolocalization of AtCPY was performed in sections of control plants (hypocotyl, A and D; root, G) or Sortin1-treated plants (hypocotyl, B and E; root, H). A preimmune serum was used as a control (C and F). Immunolocalization of invertase AtFruct4 was performed in Sortin1-treated roots (I). A–C and G–I show apoplast whereas D–F are of vacuoles. Arrowheads indicate the position of gold particles. Golgi (g) (J Left) and ER (J Right) morphology was examined in 1-week-old Sortin1-treated seedlings by electron microscopy. Tonoplast morphology in 1-week-old seedlings expressing EGFP:δ-TIP and treated with Sortin1 was analyzed by confocal microscopy (K). Arabidopsis suspension cells were treated with Sortin1 for 16 h and examined by brightfield light microscopy (L). (Scale bars = 200 nm in A–J and 20 μmin K and L.)