Results Screening for Biologically Active Compounds in Yeast. In wild-type yeast strains, CPY is delivered to the vacuole by means of Golgi-to-endosome and endosome-to-vacuole pathways and cannot be detected outside the cell. Yeast vacuolar protein sorting (vps) mutants, by contrast, secrete a significant amount of CPY ( 26). This vps phenotype resulting from treatment with specific chemicals was the basis of our screen. We screened 4,800 compounds using an anti-CPY dot-blot immunoassay for the CPY secretion phenotype in the yeast strain INVSc1, which does not secrete CPY under normal conditions. The library chosen was available commercially and was composed of a diverse set of chemical structures ( www.hit2lead.com). The library was supplied in a mg/liter format and has been used successfully by other researchers ( 27). As predicted for the screening of a library of diverse chemical structures, the preponderance of compounds (99%) had no detectable effect in initial screens. Nine compounds had fungicidal properties resulting in yeast lethality and were not examined further (data not shown). Forty compounds (0.8% of the total) were identified as “hits” in the primary screen. We confirmed that 14 (0.3% of the total) of these 40 compounds possessed biological activity at concentrations of 10 mg/liter and 100 mg/liter. The identification numbers of these 14 compounds according to the Chembridge database ( www.hit2lead.com) and a qualitative description of the resulting CPY secretion under the primary screening concentration are presented in Table 1. | Table 1. Effects of Sortins on the CPY secretion of yeast (vps phenotype) or the morphology of Arabidopsis or yeast vacuoles |
Based on the primary screen phenotype, we named these drugs “Sortins” for protein sorting inhibitors. Two of them, Sortin1 ( Fig. 1A) and Sortin2 ( Fig. 1B) were considered high-priority hits based on the significant amount of secreted CPY ( Fig. 1 D and E). Interestingly, doses of Sortin1 >25 mg/liter resulted in lesser, although still significant, stimulation of secretion. Although we do not understand the basis of this observation, the experiment was highly reproducible. The remaining 12 compounds showed a weak degree of secretion enhancement compared with Sortin1 and -2, which was typified by Sortin3 ( Fig. 1F). Untreated control cells showed little or no detectable secretion using the immunoblot assay ( Fig. 1 D–F; 0 mg/liter). In addition to the vps phenotype, yeast cells were examined for an altered vacuolar morphology ( vam) phenotype ( 28). When viewed by confocal microscopy using the specific vacuolar membrane dye MDY-64, 13 of the 14 compounds resulted in no obvious effect on yeast vacuolar morphology ( Table 1). However, one of the compounds, Sortin3 ( Fig. 1C), when compared with the untreated control cells ( Fig. 1G), induced a dramatic and severe vam phenotype in yeast ( Fig. 1H). This phenotype was similar to that of the control yeast mutant vps16Δ, which displays a vam phenotype ( 12) ( Fig. 1I). In a search of the SciFinder database (Version 2004, American Chemical Society), we found no references to compounds with the structures of Sortin1, -2, and -3 having biological activity. | 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 (more ...) |
Sortins Result in Aberrant Vacuoles in Intact Plants and Are Reversible. All fourteen compounds were tested on Arabidopsis plants expressing EGFP:δ-TIP ( 15). Seedlings were germinated and grown in the presence of the drugs for 1 week and examined for vacuolar phenotype by confocal microscopy. Interestingly, compared with untreated control plants ( Fig. 2A), the vacuolar membranes in hypocotyls of seedlings grown in the presence of 100 mg/liter (227 μM) Sortin1 or Sortin2 (233 μM), high-priority hits from the initial screen, seemed highly fragmented ( Fig. 2 B and C). These findings suggest that the compounds likely target the vacuolar biogenesis machinery in plants. The remaining 12 compounds had no effect on vacuolar morphology at 100 mg/liter tested (data not shown). | 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 (more ...) |
In addition to a tonoplast morphology phenotype, Sortin1 also severely affected root development at 100 mg/liter. Despite this dramatic effect, the phenotype induced by Sortin1 was found to be reversible after transfer to non-drug medium ( Fig. 2D). Sortin2 treatment also resulted in a dramatic inhibition of root development at 100 mg/liter and displayed reversibility similar to that of Sortin1 ( Fig. 2E). Sortin1 Stimulates Protein Secretion in Arabidopsis Suspension Cells. To examine the effect of Sortin1 and Sortin2 on secretion at the biochemical level, we treated plant suspension cells with the drugs. Cell viability was first examined at several concentrations over time in Arabidopsis and tobacco BY2 cells by using the vital dye fluorescein-diacetate (data not shown). When compared with untreated control cells ( Fig. 3 A and D), Sortin1 was well tolerated at a concentration of 25 mg/liter (57 μM) for >16 h by both Arabidopsis and tobacco cells ( Fig. 3 B and E). To the contrary, Sortin2 was highly toxic, and the cells were found without metabolic activity at a concentration of 25 mg/liter (58 μM) after 8 h ( Fig. 3 C and F). Cell death was also apparent at doses of Sortin2 as low as 10 mg/liter (23 μM) after only 4 h (data not shown). The similar results from Arabidopsis and tobacco cultured cells indicated that the toxic effect of the drugs was not specific to cells from a single plant species. Due to the high toxicity of Sortin2 on cultured cells, and thus the possibility of cell lysis, we focused on Sortin1 to ask whether a sublethal dose would result in a significant increase in overall protein secretion. | 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 (more ...) |
After treatment with 57 μM Sortin1 for 16 h, we concentrated the growth media and separated the proteins by SDS/PAGE. The protein profiles in Coomassie blue-stained gel lanes from untreated and treated Arabidopsis cells ( Fig. 3G, lanes 1 and 2, respectively) differed significantly, suggesting that Sortin1 triggered a general increase in protein secretion. AtCPY is known to undergo processing after delivery to the vacuole ( 22), so we assayed for intracellular and extracellular mature and precursor forms of AtCPY to provide direct evidence of altered fidelity of protein sorting. Indeed, when we analyzed the concentrated growth media by immunoblots with anti-CPY antibodies, we identified a precursor of AtCPY in medium from cells treated with Sortin1 ( Fig. 3G, lane 6) that was absent in an untreated control sample ( Fig. 3G, lane 5). Our data indicated that a notable amount of AtCPY precursor was diverted to the secretion pathway as a result of Sortin1 treatment. The lack of mature AtCPY in the medium indicated that AtCPY detected in the medium was probably the result of active secretion and not cell and vacuole lysis. A similar analysis of cell pellets did not reveal any obvious difference in intracellular processing of AtCPY ( Fig. 3G, lanes 3 and 4). This finding indicated that, at the sublethal dose examined, not all AtCPY was secreted. However, the proportion that was secreted was diverted at a step before proteolytic processing. This finding also pointed to the fine level of control that was possible with Sortin1 derived from the chemical screen. To demonstrate conclusively that Sortin1 induced secretion of the AtCPY precursor in metabolically active cells, we performed a pulse–chase experiment in Arabidopsis suspension cells in the presence of Sortin1. Cells were labeled with 35S-amino acids and chased with unlabeled amino acids in the presence or absence of Sortin1. The AtCPY that was secreted into the media was then immunoprecipitated by using anti-CPY antibodies and analyzed by SDS/PAGE and fluorography. Cells treated with Sortin1 displayed a rapid accumulation of a polypeptide corresponding to the unprocessed precursor of AtCPY. Compared with an untreated control in which little or no precursor was detected even after 24 h ( Fig. 3H, control), this accumulation was detectable in 3 h or less after Sortin1 treatment ( Fig. 3H, Sortin1). These results clearly established that Sortin1 stimulated secretion from cells that were metabolically active and intact. Sortin1 Stimulates Secretion in Whole Plants. To investigate the effect of Sortin1 in intact plants, we performed immunoelectron microscopy of AtCPY in 1-week-old Arabidopsis seedlings grown in the presence of the drug at a concentration of 57 μM. Compared with untreated controls ( Fig. 4 A and G), immunogold labeling detected a significant amount of AtCPY in the apoplast of hypocotyl ( Fig. 4B) and root ( Fig. 4H) tissue of treated seedlings. Consistent with the results of the immunoblots, AtCPY was also detected in vacuoles of untreated and treated seedlings ( Fig. 4 D and E, respectively), again indicating that, at sublethal doses, redirection to the secretion pathway was not complete. We also performed immunolocalization of the vacuolar invertase AtFruct4 ( 22) in root tissue of Sortin1-treated Arabidopsis seedlings. In young seedlings, AtFruct4 was previously localized to endoplasmic reticulum (ER)-derived precursor protease vesicles ( 22) and thus utilizes a different pathway than AtCPY for vacuolar targeting. Labeling of AtFruct4 was clearly observed in precursor protease vesicles of 1-week-old seedlings treated with Sortin1 ( Fig. 4I). Interestingly, no At-Fruct4 labeling in the apoplast was detected ( Fig. 4I), suggesting that Sortin1 probably targets specific protein sorting pathways. In addition, when we analyzed the concentrated growth medium from cultured Arabidopsis cells treated with Sortin1, no vacuolar invertase was detected, which is consistent with our immunolocalization and cell viability assays (data not shown). Several drugs known to affect endomembrane trafficking have significant impacts on Golgi morphology (reviewed in ref. 29). To further characterize the effect of 57 μM Sortin1 on the endomembrane system, we examined the morphology of the Golgi apparatus ( Fig. 4J Left) and ER ( Fig. 4J Right) in hypocotyls of 1-week-old seedlings by electron miscroscopy. Sortin1 treatment did not affect Golgi structure or trigger loss of cisternae. Additionally, 57 μM Sortin1 did not alter the morphology of either the ER or the central vacuole in seedlings ( Fig. 4K) or Arabidopsis suspension cells ( Fig. 4L). | 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 (more ...) |
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