Characterization of intermediates in the process of plant peroxisomal protein import

Pool, Martin; López‐Huertas, Eduardo; Baker, Alison

doi:10.1093/emboj/17.23.6854

Cited by 25 publications

(16 citation statements)

References 45 publications

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“…Different experimental systems have been used by several groups to address this issue. The unanimous conclusion that emerged from these studies is that energy provided by ATP hydrolysis is necessary for peroxisomal protein import into the organelle matrix (13)(14)(15)(16)(17)(18)(19)(20)(21). However, the exact steps in the Pex5p-mediated import pathway where energy input is necessary have remained unclear.…”

Section: Discussionmentioning

confidence: 99%

“…One obvious property of such cyclic mechanism is that it needs some form of energy input to function, and indeed, basically all the studies addressing this issue are unanimous in this respect: protein import into the peroxisomal matrix requires hydrolysis of ATP (13)(14)(15)(16)(17)(18)(19)(20)(21). However, the precise step(s) of this import pathway where energy input is necessary has not been firmly established.…”

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confidence: 99%

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The Energetics of Pex5p-mediated Peroxisomal Protein Import

Oliveira

Gouveia

Pinto

et al. 2003

Journal of Biological Chemistry

100

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Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system.Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally imported into the organelle. The vast majority of these proteins possess the so-called peroxisomal targeting sequence 1 (PTS1), 1 a C-terminal tripeptide complying to the consensus sequence S/A/C-K/R/H-L/M (1-4). These PTS1-containing proteins are specifically targeted to the organelle matrix by Pex5p, the PTS1 receptor (5-9).According to current models of peroxisomal biogenesis, the Pex5p-mediated process of protein import can be divided into four steps. In the first step, newly synthesized PTS1-containing proteins interact with Pex5p in the cytosol. This protein-protein interaction involves the PTS1 signal on one side and the tetratricopeptide repeats domain of Pex5p on the other. The Pex5p-cargo protein complex is then recognized by the so-called docking machinery present in the peroxisomal membrane. Somewhere after this event, the PTS1-containing protein is released into the peroxisomal matrix. Finally, Pex5p is recycled back to the cytosol to catalyze additional rounds of transportation (reviewed in Refs. 10 -12).One obvious property of such cyclic mechanism is that it needs some form of energy input to function, and indeed, basically all the studies addressing this issue are unanimous in this respect: protein import into the peroxisomal matrix requires hydrolysis of ATP (13-21). However, the precise step(s) of this import pathway where energy input is necessary has not been firmly established. For instance, it is generally accepted that the step of protein translocation across the peroxisomal membrane requires ATP hydrolysis. Such conclusion derives from the fact that ATP depletion or the inclusion of non-hydrolysable ATP analogues in the several experimental systems used result in an inhibition of the peroxisomal import process. However, the possibility that recycling of Pex5p back to the cytosol is an ATP-dependent event and the rate-limiting step in all this process was never considered. In this scenario, inhibition of peroxisomal protein import by lack of ATP would resu...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The Energetics of Pex5p-mediated Peroxisomal Protein Import

Oliveira

Gouveia

Pinto

et al. 2003

Journal of Biological Chemistry

100

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“…In this report, we used an established in vitro import assay (Brickner et al, 1997;Brickner and Olsen, 1998;Crookes and Olsen, 1998;Pool et al, 1998) to study the mechanisms of PTS2 protein import into peroxisomes. We successfully and consistently reconstituted PTS2 protein import into isolated pumpkin glyoxysomes.…”

Section: Discussionmentioning

confidence: 99%

“…It has recently been shown that human Pex5p transports a cargo PTS1 protein to the peroxisomal membrane, where it appears to enter, at least partially, the matrix of the organelle along with the PTS1 protein, and then it is recycled back to the cytosol after releasing the cargo protein in the peroxisome matrix (Dammai and Subramani, 2001;Kunau, 2001). The use of in vitro systems to examine the specifics of PTS1 import has revealed additional mechanistic information (Horng et al, 1995;Brickner et al, 1997;Brickner and Olsen, 1998;Crookes and Olsen, 1998;Fransen et al, 1998;Pool et al, 1998). This approach was used to demonstrate the time, temperature, and energy dependence of PTS1 protein import (Behari and Baker, 1993;Brickner et al, 1997;Brickner and Olsen, 1998), as well as the role of cytosolic chaperones and membrane-associated proteins in the pathway (Crookes and Olsen, 1998;Fransen et al, 1998).…”

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confidence: 99%

Import of the Peroxisomal Targeting Signal Type 2 Protein 3-Ketoacyl-Coenzyme A Thiolase into Glyoxysomes

Johnson

Olsen

2003

Plant Physiology

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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.The enzyme 3-ketoacyl-CoA-thiolase (EC 2.3.1.16; thiolase) is a homodimer that catalyzes the terminal reaction of fatty acid ␤-oxidation in eukaryotes. In many organisms, thiolase is located in both the mitochondrial matrix and the peroxisome, but in plants, it appears to be localized exclusively to peroxisomes, including the specialized type of plant peroxisome, the glyoxysome (Kindl, 1992). No thiolase activity has been localized to plant mitochondria. Like all peroxisomal matrix proteins, thiolase is synthesized on free polyribosomes in the cytosol and posttranslationally imported into the organelle (Johnson and Olsen, 2001).Most peroxisomal matrix proteins use a carboxyterminal tripeptide targeting signal, termed a peroxisomal targeting signal type 1 (PTS1; Gould et al., 1989). In contrast, thiolase is directed to the organelle by a distinct signal, the peroxisomal targeting signal type 2 (PTS2; Gietl, 1990;Osumi et al., 1991;Swinkels et al., 1991; Kato et al., 1996). The PTS2 is nonapeptide signal with the consensus sequence (R) X 6 (H/ Q)(L/A/F); it is located near the amino terminus of the peroxisomal protein (Flynn et al., 1998;Olsen, 1998). In addition to thiolase, other plant proteins that contain a PTS2 include citrate synthase (Kato et al., 1996), malate dehydrogenase (Gietl et al., 1994), amine oxidase (Faber et al., 1995), and at least one isozyme of Asp aminotransferase (Gebhardt et al., 1998). Plants appear to have more PTS2 proteins than other organisms.Relatively few proteins follow the PTS2 pathway, but their import is necessary for the survival of the organ...

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“…An important technical handicap to further progress is the lack of an in vitro reconstituted protein-import system similar to those for mitochondria, ER and chloroplasts. Recently, some hope was raised by successful reconstitution steps using plant glyoxysomes and protein constructs that remain jammed in the import site 25 .…”

Section: Figurementioning

confidence: 99%