A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways

Abstract

SUMMARYThe use of small molecules has great power to dissect biological processes. This study presents the identification and characterisation of an inhibitor of peroxisome matrix protein import. A mini-screen was carried out to identify molecules that cause alteration in peroxisome morphology, or mislocalization of a peroxisome targeted fluorescent reporter protein. A benzimidazole lead compound (LDS-003655) was identified that resulted in reduced GFP fluorescence in peroxisomes and cytosolic GFP accumulation. The effect of the compound was specific to peroxisomes as Golgi bodies, endoplasmic reticulum and the actin cytoskeleton were unaffected even at 25 lM, whereas peroxisome import via the PTS1 pathway was compromised at 100 nM. When seedlings were grown on 25 lM LDS-003655 they displayed morphology typical of seedlings grown in the presence of auxin, and expression of the auxin reporter DR5::GFP was induced. Analysis of a focussed library of LDS-003655 derivatives in comparison with known auxins led to the conclusion that the auxin-like activity of LDS-003655 is attributable to its in situ hydrolysis giving rise to 2,5-dichlorobenzoic acid, whereas the import inhibiting activity of LDS-003655 requires the whole molecule. None of the auxins tested had any effect on peroxisome protein import. Matrix import by the PTS2 import pathway was relatively insensitive to LDS-003655 and its active analogues, with effects only seen after prolonged incubation on high concentrations. Steady-state protein levels of PEX5, the PTS1 import pathway receptor, were reduced in the presence of 100 nM LDS-003655, suggesting a possible mechanism for the import inhibition.

PEX14 binding to Arabidopsis PEX5 has differential effects on PTS1 and PTS2 cargo occupancy of the receptor

“…Immunoblotting was as described [30] using anti‐ Arabidopsis PEX5 [29] at 1:10 000 dilution, anti‐ Arabidopsis PEX7 [31] at 1:1000 dilution, and anti‐thiolase [32] at 1:180 000 dilution.…”

Plant Peroxisomes: Biogenesis and Function

“…Alternative approaches, such as chemical genetics, can be valuable in the dissection of essential processes (Hicks and Raikhel, 2009). A group of benzimidazole compounds have been identified to differentially disrupt PTS1 protein import at nanomolar concentrations, whereas PTS2 import inhibition is only seen after long incubation at micromolar concentrations (Brown et al, 2011). Protein import in general is poorly understood at the mechanistic level, and kinetic and quantitative data on protein interactions within the import pathway would help in building and testing models.…”

An inhibitor of oil body mobilization in Arabidopsis

“…Diphenyl methylphosphonate treated seedlings retain oil bodies but other organelles are unaffected A small scale confocal laser scanning microscopy (CLSM)-based screen for compounds that altered peroxisome morphology and/or fluorescence intensity in an Arabidopsis line that expresses a peroxisomal targeted GFP reporter was carried out (Brown et al, 2011). The 70 compounds used in the small scale screen originated from a primary screen for compounds that affected hypocotyl gravitropism (Surpin et al, 2005) thus were known to be bioavailable and bioactive in plants.…”