Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes

Abstract

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.

Figure 1.

Alignment of 3-ketoacyl-CoA thiolase from Arabidopsis and canola. The full-length amino acid sequences of 3-ketoacyl-CoA thiolase from Arabidopsis (At; accession no. T20943) and canola (Bn; accession no. CAA63598) are shown. These two proteins differ in only 17/462 residues. The PTS2 import signal is underlined. The Ser-rich region found in many PTS2 proteins is indicated in bold. The Leu in the full-length protein at the predicted cleavage site of the PTS2 processing protease is indicated by a black box; this Leu was changed to a Met as the first residue of the engineered mature thiolase protein.

Figure 2.

In vitro import of GLO, full-length thiolase (THL), and mature thiolase (mTHL) into isolated pumpkin glyoxysomes. Isolated glyoxysomes were incubated with radiolabeled GLO (top) for 30 min, full-length thiolase (middle) for 60 min, or mature thiolase (bottom) for 60 min under standard import conditions (see “Materials and Methods”) at either 25°C (lanes 1-3) or 4°C (lane 4). After import, thermolysin was added to samples in lanes 2 to 4. The detergent Triton X-100 was added to samples in lane 3 to lyse the organelles. All subsequent treatments were as described in “Materials and Methods.” The results shown are from representative experiments that were repeated three times for GLO and mature thiolase and four times for full-length thiolase. TR, Translation product.

Figure 3.

Import of thiolase (THL) into glyoxysomes is slower than import of GLO. Standard in vitro import reactions were performed for 0 to 180 min for thiolase (solid line) and for 0 to 120 min for GLO (dotted line), as described in “Materials and Methods.” At the indicated time points, samples were placed on ice to stop the import reactions and treated with thermolysin to remove nonimported proteins. The level of GLO import at 40 min and the amount of thiolase imported at 180 min were set at 100% relative import for comparison with the other time points from the same experiment. Each protein reached a maximum import level of approximately 10% of the radiolabeled protein presented to the organelles. The experiment was repeated six times with thiolase and four times with GLO; the results are presented as the average ± se.

Figure 4.

Import of thiolase is decreased by excess thiolase but not by the PTS1 protein GLO. Isolated glyoxysomes were incubated with increasing amounts of non-radiolabeled full-length thiolase (A) or GLO (B) in the presence of a constant amount of radiolabeled protein. The amount of trichloroacetic acid-precipitable counts obtained from a standard translation reaction was used to calculate the equivalent amount of non-radiolabeled protein for import reactions. For example, twice as much non-radiolabeled as radiolabeled protein is “2 equivalents,” and so on. The amount of thiolase imported in the absence of non-radiolabeled protein was set at 100% for comparison with the other data points from this experiment. The experiments were repeated three times; the results are presented as the average ± se.

Figure 5.

Import of full-length thiolase, but not GLO, is inhibited by mature thiolase. A, Isolated glyoxysomes were incubated with increasing amounts of radiolabeled mature thiolase in the presence of a constant amount of radiolabeled full-length thiolase protein. The amount of protein imported in the absence of radiolabeled mature thiolase was set at 100% for comparison with the other data points from this experiment. B, Isolated glyoxysomes were incubated with increasing amounts of radiolabeled mature thiolase in the presence of a constant amount of radiolabeled GLO. The amount of GLO imported in the absence of mature thiolase was set at 100% for comparison with the other data points from this experiment. The experiments were repeated three times; the results are presented as the average ± se.

Figure 6.

Import of thiolase (THL) is stimulated by the addition of zinc chloride. Isolated glyoxysomes were incubated with increasing amounts of zinc chloride or the zinc chelator, 1-10-phenanthroline, in the presence of a constant amount of radiolabeled thiolase. The amount of thiolase imported in the absence of zinc chloride was set at 100% for comparison with the other data points from the experiment. The experiments were repeated three times; the results presented represent the average ± se.

Figure 7.

Zinc stimulates PTS2 protein import. Isolated glyoxysomes were incubated with different concentrations of zinc or potassium salts in the presence of a constant amount of radiolabeled thiolase. The amount of thiolase imported in the absence of additional salt (black bar) was set at 100% for comparison with the other data points from each experiment. The salts added were zinc chloride and potassium chloride (light gray bars), zinc acetate (ZnAc₂) and potassium acetate (KAc₂; dark gray bars), and zinc sulfate and potassium sulfate (white bars). The experiments were repeated three times; the results presented represent the average ± se.

Figure 8.

In vitro binding of thiolase (THL) is increased in the presence of zinc. Isolated glyoxysomes were incubated with radiolabeled thiolase (THL) or mature thiolase (mTHL) at either 25°C (lanes 1 and 2) or 4°C (lanes 3 and 4). Zinc was added to samples in lanes 2 and 4. All subsequent treatments were as described in “Materials and Methods.” None of the samples was treated with protease. The results shown are from representative experiments that were repeated three times with mature thiolase and four times with full-length thiolase.