Nucleotide triphosphates are required for the transport of glycolate oxidase into peroxisomes

Abstract

All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

Figure 2

Import of GLO into isolated glyoxysomes requires Mg²⁺. Standard import reactions were performed as described in Figure 1 and Methods except that 5 mm Na₂ATP was present in each reaction instead of MgATP. All other import reactions were supplemented with additional cations (supplied by MgCl₂, MnCl₂, CaCl₂, or KCl) at 5 mm final concentration. The level of import observed with 5 mm MgCl₂ (and 5 mm Na₂ATP) added to the import reaction was set at 100% for comparison with import levels in the presence of the other cations. The values presented are the average ± se of two experiments.

Figure 7

Ionophores reduce the level of glyoxysomal protein import. To determine whether a PMF plays a role in peroxisomal protein import, isolated glyoxysomes were preincubated for 20 min at room temperature with various ionophores (1–20 μm final concentration). Import reactions were performed and analyzed as described in Figure 1. The level of protease-resistant GLO present in samples that had no ionophore added was set as 100% relative import for comparison with the ionophore-treated samples. A representative experiment is presented. A, GLO import into glyoxysomes in the presence of nigericin or valinomycin, ionophores that collapse a single component of the PMF. B, GLO import into glyoxysomes in the presence of ionophores that collapse the total PMF.

Figure 1

Import of GLO into glyoxysomes is energy dependent. To characterize the NTP dependence of protein transport, increasing concentrations of ATP (○) or GTP (•) were added to standard import reactions (see Methods). Before addition to the import reactions, GLO translation products were desalted on a Sephadex G-25 column to remove endogenous nucleotides and other small molecules. The amount of radiolabeled GLO that remained protease protected after import in the presence of 5 mm ATP was set as 100% relative import for comparison with the other samples. The average ± se of three independent experiments is shown.

Figure 3

Nonhydrolyzable ATP analogs cannot support GLO import. To determine whether ATP hydrolysis is required for peroxisomal protein import, increasing amounts of ATP, AMP-PCP, or AMP-PNP (nonhydrolyzable analogs of ATP) were added to standard import reactions. Before addition to the import reactions, GLO translation products were desalted on a Sephadex G-25 column to remove endogenous nucleotides and other small molecules (see Methods). The amount of GLO imported into glyoxysomes in the presence of 5 mm ATP was set at 100% for comparison with the other samples in the same experiment. The average ± se of three independent experiments is shown.

Figure 4

GTP hydrolysis is required for peroxisomal protein import. To determine whether the hydrolysis of GTP was required for peroxisomal protein import, increasing amounts of either GTP or GTP-γ-S were added to NTP-depleted import reactions, as described in Figure 1. The average ± se of two independent experiments is shown.

Figure 5

AMP-PCP competes with ATP during protein import. Isolated glyoxysomes were incubated with increasing amounts of ATP and challenged with the nonhydrolyzable ATP analog, AMP-PCP, at the concentrations indicated. Subsequent import reactions were performed as described in Figure 1. The results shown are the average ± se of three separate experiments.

Figure 6

GTP-γ-S inhibits ATP-dependent protein import. To examine the effects of GTP-γ-S on ATP-dependent import, ATP and/or excess GTP-γ-S were preincubated with isolated glyoxysomes for 5 min. Protein import was initiated by the addition of radiolabeled GLO proteins. The amount of radiolabeled GLO imported in the presence of 5 mm ATP was set as 100% relative import. Because of minor differences in the ways in which each replicate experiment was performed, a representative experiment is presented.