Plant Physiol. (1998) 116: 309–317
Nucleotide Triphosphates Are Required for the Transport of
Glycolate Oxidase into Peroxisomes1
Donna G. Brickner and Laura J. Olsen*
Department of Biology, University of Michigan, Ann Arbor, Michigan 48109–1048
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 Mg21 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.
Peroxisomes are ubiquitous organelles involved in a variety of important cellular processes, including the degradation of hydrogen peroxide and the b-oxidation of fatty
acids (for review, see Olsen and Harada, 1995). The single
peroxisomal membrane surrounds a dense matrix, sometimes containing paracrystalline inclusions (Frederick et
al., 1975). New organelles are thought to arise by the fission
of preexisting peroxisomes, with the subsequent incorporation of additional membrane lipids and peroxisomal proteins. There are several classes of peroxisomes found in
higher plants (Kindl, 1992; Gietl, 1996). Glyoxysomes are
abundant in the cotyledons of most plants and are involved
in lipid mobilization to provide nutrients during germination and seedling growth (Trelease, 1984). In leaves, peroxisomes contain enzymes such as GLO that are necessary
for photorespiration (Ogren, 1984).
All peroxisomal proteins must be nuclear encoded because peroxisomes do not contain DNA. Peroxisomal proteins are synthesized on free, cytosolic ribosomes and im-
ported posttranslationally into the organelle. The majority
of matrix proteins, including GLO, are targeted to peroxisomes via a carboxyl-terminal tripeptide comprising the
amino acids Ser-Lys-Leu or conserved variants (Gould et
al., 1987; McNew and Goodman, 1996). Other peroxisomal
proteins, such as malate dehydrogenase and thiolase, use
an amino-terminal targeting signal (Gietl, 1990; Swinkels et
al., 1991; Preisig-Müller and Kindl, 1993). Considerable
genetic and biochemical evidence indicates the involvement of a proteinaceous receptor in peroxisomal protein
import (Wolins and Donaldson, 1994; Rachubinski and
Subramani, 1995; Dodt and Gould, 1996; McNew and
Goodman, 1996; Brickner et al., 1997). However, a detailed
description of the targeting mechanism and an understanding of receptor localization remain elusive. Protein import
into yeast peroxisomes may also use other proteins (Dodt
and Gould, 1996), including a cytosolic ATPase (Yahraus et
al., 1996), a membrane-bound docking factor (Elgersma et
al., 1996; Erdmann and Blobel, 1996; Gould et al., 1996), an
N-ethylmaleimide-sensitive factor (Wendland and Subramani, 1993), and a molecular chaperone such as hsp70
(Walton et al., 1994).
Protein trafficking requires the investment of energy.
The hydrolysis of ATP and/or GTP is required for protein
transport into mitochondria (Pfanner et al., 1990), chloroplasts (Keegstra et al., 1989; Theg et al., 1989; Kessler et al.,
1994), nuclei (Powers and Forbes, 1994), and the ER (Walter
and Johnson, 1994). ATP hydrolysis is known to be necessary for peroxisomal protein import (Imanaka et al., 1987;
Wendland and Subramani, 1993; Horng et al., 1995; Brickner et al., 1997). A careful analysis of the energy requirements for protein binding to chloroplasts revealed that
other NTPs can support this process, although less efficiently than ATP (Olsen et al., 1989). Exactly where and
how the energy is being used during translocation is currently unknown. It is likely that NTP hydrolysis induces
conformational changes in the translocation machinery or
in the targeted protein (perhaps by interaction with an
energy-dependent molecular chaperone), thereby facilitating the protein’s entry into the organelle (Pfanner et al.,
1990).
A PMF is required, in addition to ATP, for the export of
proteins from bacteria (Yamane et al., 1987; Wong and
1
This work was funded by a grant from the U.S. Department of
Agriculture to L.J.O. D.G.B. was supported in part by a fellowship
from the Cellular Biotechnology Training Program (National Institutes of Health grant no. GM08353).
* Corresponding author; e-mail [email protected]; fax 1–734 – 647–
0884.
Abbreviations: A23187, calcinomycin; AMP-PCP, methylene
adenosine 59-triphosphate; AMP-PNP, 59-adenylylimidodiphosphate; CCCP, carbonyl cyanide m-chloro-phenylhydrazone; GLO,
glycolate oxidase; NTP, nucleotide triphosphate; PMF, protonmotive force.
309
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310
Brickner and Olsen
Buckley, 1989); a pH gradient is needed to transport some
proteins across thylakoid membranes (Cline et al., 1992;
Theg and Scott, 1993); and the electrical component of the
PMF facilitates the import of some proteins into mitochondria (Pfanner and Neupert, 1986). Addition of inhibitors or
ionophores that collapse the PMF abolishes protein transport across each of these membranes. A PMF may exist
across the peroxisomal membrane. Some researchers have
found that ionophores inhibit protein import into peroxisomes (Bellion and Goodman, 1987), whereas others observed no effect on peroxisomal protein import (Imanaka et
al., 1987; Wendland and Subramani, 1993). An ATPase,
analogous to the V-class H1-ATPase found on vacuolar
membranes, may be present on the peroxisomal membrane
(Douma et al., 1987; del Valle et al., 1988; Wolvetang et al.,
1990; Whitney and Bellion, 1991). ATP hydrolysis on the
cytosolic face would make the peroxisomal matrix acidic
(Nicolay et al., 1987), thus establishing a PMF that could be
used to drive protein translocation. Alternatively, nonspecific pores may allow small ions and metabolites to diffuse
freely across the membrane, thereby dissipating an electrochemical or pH gradient (for review, see van den Bosch et
al., 1992).
We have extensively characterized the energy requirements for protein import into glyoxysomes using an optimized in vitro assay to reconstitute the transport event.
First, we examined the ability of various NTPs to support
the import of GLO into isolated pumpkin (Cucurbita pepo)
glyoxysomes. The competence of nonhydrolyzable analogs
of ATP and GTP to substitute for the NTP requirement was
also analyzed. Next, we briefly investigated the cation
specificity of the nucleotide requirement for protein import. Finally, in an effort to characterize the role that a
putative PMF might play, we explored the effect of ionophores on GLO import. Ionophores were chosen that collapsed the pH gradient, the electrical gradient, or both
components of the PMF. A thorough study of the energetics of protein import into peroxisomes is necessary to facilitate the understanding of the molecular mechanisms
involved in the transport process.
MATERIALS AND METHODS
All NTPs, NTP analogs, ionophores, and general chemicals were purchased from Sigma. Sephadex G-25–80 (fine)
was purchased from Pharmacia. Pumpkin (Cucurbita pepo
var Half Moon) seeds were purchased from Petoseed Co.,
Inc. (Saticoy, CA). Redi-vue [35S]Met (specific activity, 43.5
TBq/mmol) was purchased from Amersham.
Preparation of Radiolabeled GLO
The plasmid pGLOZf, containing a cDNA insert for the
entire coding region of the peroxisomal enzyme GLO in the
pGEM7Zf(1) vector (Promega), was linearized with HindIII and transcribed with SP6 RNA polymerase as described by Brickner et al. (1997). Radiolabeled GLO was
synthesized in a cell-free, wheat germ lysate system in the
presence of [35S]Met. To test the NTP requirements for
protein import, free nucleotides were removed from the
Plant Physiol. Vol. 116, 1998
translation products by size-exclusion chromatography using Sephadex G-25–80 (fine) as described by Olsen et al.
(1989). The efficiency of the translation reaction was assessed by TCA precipitation of the proteins onto glass fiber
filters, followed by ethanol washes and quantitation in a
liquid scintillation counter (model LS 6800, Beckman).
Standard import reactions contained radiolabeled GLO
equivalent to 0.5 3 106 to 1.0 3 106 TCA-precipitable
counts (usually 3–15 mL).
Isolation of Pumpkin Glyoxysomes
Pumpkin seeds were germinated in damp vermiculite for
5 to 6 d at 28 to 30°C in complete darkness. For each
experiment approximately 40 g of cotyledons was harvested manually in dim light. Glyoxysomes were isolated
as described by Brickner et al. (1997). The organelle isolation buffer included 10 mm azide for all experiments except
the cation specificity and the ionophore experiments (see
below).
In Vitro Import Assays
Standard import reactions contained glyoxysomes (80–
500 mg of protein), radiolabeled GLO, 5 mm ATP (Mg21
salt), and import buffer (25 mm Mes-KOH, pH 6.0, 0.5 m
Suc, 10 mm KCl, 1 mm MgCl2, sometimes also with 10 mm
NaN3; see below) in a final volume of 200 mL. All import
reactions were initiated by the addition of translation products and incubated at 26°C for 30 min. After import, samples were treated with 10 mg/mL proteinase K for 30 min
on ice to digest translation products not protected by the
glyoxysomal membrane. Protease treatment was stopped
by the addition of the inhibitor PMSF (1 mm final concentration). Intact glyoxysomes were reisolated on a 0.7 m Suc
cushion and centrifuged at 8500g for 15 min in a refrigerated microcentrifuge. The pellets were resuspended in
SDS-PAGE sample buffer, heated at 80 to 90°C for 2 to 5
min, and stored at 220°C until further analysis. Radioactive proteins were analyzed by SDS-PAGE and visualized
by fluorography.
The Mg21 salt of ATP was used in most standard import
reactions, except that Na2ATP was used for the cation
specificity experiments (see Fig. 2). Other import reactions
were supplemented with additional cations as indicated in
the legend to Figure 2. Import reactions containing other
NTPs and/or NTP analogs were supplemented with
equimolar MgCl2 in addition to the 1 mm MgCl2 present in
the import buffer. The presence of Mg21 by itself had no
effect on import (data not shown).
Glyoxysomes were preincubated with ionophores and 5
mm ATP for 20 min at room temperature before radiolabeled GLO was added to start the import reaction. The
endogenous NTPs were not removed from the radiolabeled
GLO used in the ionophore experiments. Modified import
buffer (containing only 25 mm Mes-KOH, pH 6.0, and 0.5 m
Suc; i.e. lacking the 10 mm KCl, 1 mm MgCl2, and azide)
was used in the cation specificity experiments.
Azide (10 mm NaN3) was included in the organelle isolation buffer and import buffer in all experiments except
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Energetics of Peroxisomal Protein Import
the cation specificity (see Fig. 2) and the ionophore experiments (see Fig. 7). The presence of azide during organelle
isolation and in import reactions did not affect import
levels (data not shown). The purpose of the azide was to
inhibit any ATP synthesis by the small amounts of mitochondria that may have been present in the glyoxysome
preparation (Brickner et al., 1997), and thus remove any
endogenous ATP supplied by the mitochondria. Azide was
not included in the cation-specificity and ionophore experiments because the exact level of ATP present was not
critical. In addition, we wanted to control precisely the
amount of Na present in the import reactions when testing
for cation effects on import.
Each of the ionophore stock solutions was prepared in
ethanol; dilutions of the stocks were used in the import
reactions presented in Figure 7. Ethanol by itself, equivalent to the highest concentration present in the ionophore
treatments (approximately 0.4%), had no effect on the level
of GLO import (data not shown). Import reactions testing
the effect of the Ca21 ionophore A23187 also contained 10
mm CaCl2; 10 mm CaCl2 was also present in the controls
and all import samples testing the Ca21 ionophore A23187;
10 mm KCl was included in the controls and import samples testing valinomycin.
Quantitation
Levels of protein import were quantified by rehydrating
manually excised, radioactive gel slices in 30% hydrogen
peroxide overnight at 50°C. Standard scintillation cocktail
for aqueous samples was added and samples were counted
on a liquid scintillation counter (LS 6800, Beckman). Unless
noted otherwise, the amount of protease-protected GLO
detected after 30 min of import in the presence of 5 mm
ATP was set at 100% relative import for comparison with
the other treatments. We routinely got import efficiencies
of 5 to 15% of the added radiolabeled protein imported into
the isolated glyoxysomes in control samples. This corresponds to an average import of 3.2 3 105 molecules
GLO/mg of glyoxysomes. The actual numbers for the experiments presented here are as follows: Table I, 5 mm ATP
control, 3.4 3 105 molecules GLO/mg glyoxysomes; Figure
1, 5 mm ATP control, 3.2 3 105 molecules GLO/mg glyoxysomes; Figure 2, 5 mm Mg21 supplementing the 5 mm
ATP(Na1) salt, 5.8 3 105 molecules GLO/mg glyoxysomes;
Figure 3, 5 mm ATP control, 6.2 3 105 molecules GLO/mg
glyoxysomes; Figure 4, 5 mm GTP control, 1.9 3 105 molecules GLO/mg glyoxysomes; Figure 5, 5 mm ATP control,
1.7 3 105 molecules GLO/mg glyoxysomes; Figure 6, 5 mm
ATP control, 1.4 3 105 molecules GLO/mg glyoxysomes;
and Figure 7, no-ionophore control, 1.8 3 105 molecules
GLO/mg glyoxysomes.
RESULTS
NTP Requirement for Peroxisomal Protein Import
The energy requirements for the import of the leaf peroxisomal protein GLO were assessed using an in vitro
import assay system described by Brickner et al. (1997).
311
Glyoxysomes were isolated from dark-grown pumpkin
cotyledons and incubated with radiolabeled GLO at 26°C
for 30 min in the presence of various NTPs. The amount of
protein imported into the organelle was measured by protease resistance; all protease-resistant GLO was assumed to
be protected by the peroxisomal membrane.
As shown in Figure 1, the amount of GLO imported
increased with higher concentrations of ATP or GTP. Maximal levels of import were observed at 5 mm ATP; no
additional GLO was imported in the presence of 10 mm
ATP (Brickner et al., 1997). Low levels of protein import
(approximately 18–22%, relative to import at 5 mm ATP)
were observed even when no exogenous NTP was added to
the import reaction. This background level of import has
been observed by others (Imanaka et al., 1987; Horng et al.,
1995) and may be mediated by NTP bound to a component
of the isolated organelles, to translation products, or to a
cytosolic factor present in the wheat germ lysate; free NTPs
were removed by the desalting procedure (Olsen et al.,
1989). GTP supported GLO import nearly as well as ATP.
Maximal GTP-dependent import was achieved at a lower
NTP concentration; only 1 mm GTP was needed, compared
with 5 mm ATP. This result suggests that GTP itself can be
used as an energy source for protein import. It does not
distinguish between a single energy-dependent process,
which can use either ATP or GTP to drive import, and two
separate energy-requiring steps, one dependent on ATP
and the other dependent on GTP. Finally, the addition of
both ATP and GTP to the same import reaction stimulated
import only slightly more than the level in the control
samples (data not shown).
In addition to ATP and GTP, other NTPs were assessed
for their ability to support peroxisomal protein import
Figure 1. Import of GLO into glyoxysomes is energy dependent. To
characterize the NTP dependence of protein transport, increasing
concentrations of ATP (E) or GTP (F) were added to standard import
reactions (see “Materials and 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 6 SE of three independent experiments is shown.
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312
Brickner and Olsen
Table I. NTPs are capable of supporting import of GLO into isolated glyoxysomes
Standard import assays were performed as described in Figure 1,
except that each reaction contained 5 mM NTP, as indicated. The
values presented are the average (6 SE) of three experiments.
NTP
Percent Relative Importa
None
ATP
GTP
CTP
UTP
TTP
ITP
22 6 4
100
83 6 11
76 6 12
77 6 11
51 6 3
27 6 4
a
The import level observed with 5 mM ATP was set at 100% for
comparison with the other NTPs.
(Table I). Standard import reactions were performed in the
presence of 5 mm NTP; the radiolabeled GLO translation
products were desalted to remove ATP added to the translation reaction or present in the wheat germ lysate (see
“Materials and Methods”). With the exception of ITP, all
NTPs tested in our in vitro protein import system could
serve as an alternative energy source for peroxisomal protein import, although much less efficiently than ATP.
Cation Specificity for Peroxisomal Protein Import
Standard import reactions were performed in the presence of 5 mm ATP, provided as the Mg21 salt. The disodium salt of ATP did not support GLO import unless
equimolar levels of MgCl or Mg21 acetate were also added
to the reactions (Fig. 2). There was no difference in the level
Figure 2. Import of GLO into isolated glyoxysomes requires Mg21.
Standard import reactions were performed as described in Figure 1
and “Materials 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
6 SE of two experiments.
Plant Physiol. Vol. 116, 1998
of protein import in reactions containing the Mg21 salt of
ATP compared with reactions containing disodium ATP
supplemented with equimolar Mg21 salts (data not
shown); Mg21 alone, i.e. without ATP, had no effect on
import (data not shown). To determine whether other cations could substitute for Mg21 during protein import, various salts were added to import reactions in the presence of
disodium ATP. As seen in Figure 2, import in the presence
of the divalent cations Mn21 and Ca21 was slightly better
than with Na alone, but was still significantly less than
import in reactions supplemented with Mg21. K1 did not
increase protein import at all above the disodium ATP
background level.
NTP Hydrolysis Is Required for Peroxisomal
Protein Import
Hydrolysis of the high-energy bonds of ATP may cause
a conformational change in a component of the translocation machinery or in the translocating protein itself to
facilitate protein entry into the organelle. To determine
whether the hydrolysis of ATP is required for peroxisomal
protein import, we incubated isolated glyoxysomes and
radiolabeled GLO with the nonhydrolyzable ATP analogs
AMP-PCP and AMP-PNP. Figure 3 shows that these nonhydrolyzable analogs of ATP did not substitute as an energy source for glyoxysomal protein import. Thus, ATP
hydrolysis is required at some step during glyoxysomal
protein import.
Because GTP was also capable of supporting peroxisomal protein import (Fig. 1), the necessity of GTP hydrolysis
was examined. GTP-g-S, a nonhydrolyzable analog of GTP,
was added to standard import reactions in the absence of
other NTPs, but was unable to increase the import of GLO
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 AMPPNP (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 “Materials
and 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 6 SE of three
independent experiments is shown.
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Energetics of Peroxisomal Protein Import
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 GTPg-S were added to NTP-depleted import reactions, as described in
Figure 1. The average 6 SE of two independent experiments is shown.
to greater than background levels (Fig. 4). Therefore, it
appears that the hydrolysis of GTP may also provide energy to drive peroxisomal proteins across the membrane.
Two components of the chloroplast protein import machinery have been shown to bind GTP (Kessler et al., 1994).
There are at least two proteins involved in peroxisome
biogenesis or function that have ATP-binding domains
(Swartzman et al., 1996; Yahraus et al., 1996). One approach
to the determination of whether the NTP hydrolysis requirement for peroxisomal protein import is caused by
direct binding of the NTP by a protein factor (rather than
an indirect metabolic role for NTP) is the use of nucleotide
competition experiments. For these experiments an increasing concentration of the nonhydrolyzable ATP analog
AMP-PCP was preincubated with ATP and isolated
glyoxysomes. The import reaction was then initiated by the
addition of radiolabeled GLO protein. As shown in Figure
5, the amount of protease-protected GLO decreased with
increasing amounts of nonhydrolyzable ATP analog even
in the presence of ATP. This competitive interaction be-
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 6
SE of three separate experiments.
313
tween ATP and AMP-PCP indicates that there may be a
discrete nucleotide binding site on some component of the
translocation apparatus, or on another factor that is required for import of proteins into peroxisomes.
To assess the effect of a nonhydrolyzable GTP analog on
ATP-dependent protein import levels, NTP competition
experiments were performed using GTP-g-S as the competitor. Competition was established by challenging an import
reaction containing ATP with an excess of GTP-g-S (Fig. 6).
The addition of 10 mm GTP-g-S significantly reduced the
level of GLO imported into glyoxysomes in the presence of
5 mm ATP. This suggests that ATP and GTP are either
competing for a common nucleotide binding site on some
component required for protein translocation, or that both
NTPs are required at different steps of protein import, one
of which is inhibited by the binding of GTP-g-S.
Effects of Ionophores on Peroxisomal Protein Import
To determine whether a PMF contributes to protein import, a variety of ionophores was added to standard in
vitro import reactions. Isolated glyoxysomes were preincubated for 20 min at room temperature with either nigericin
(a H1/K1 antiporter) to collapse the pH gradient, valinomycin (a K1 uniporter) to dissipate the membrane potential, or gramicidin (a channel former), CCCP (a H1
uniporter), A23187 (a Ca21/H1 antiporter), or nigericin
and valinomycin together to collapse both components of
the PMF. Each ionophore reduced the level of GLO import
to roughly 50 to 60% of the control import reaction when
present at 10 to 20 mm (Fig. 7). Nigericin and valinomycin
are typically used at micromolar to submicromolar concentrations (Pfanner and Neupert, 1986; Nicolay et al., 1987;
Cline et al., 1992; Theg and Scott, 1993). In our experiments,
low concentrations (1 and 2 mm) of nigericin and valinomycin had little or no effect on peroxisomal protein import
Figure 6. GTP-g-S inhibits ATP-dependent protein import. To examine the effects of GTP-g-S on ATP-dependent import, ATP and/or
excess GTP-g-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.
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314
Brickner and Olsen
Plant Physiol. Vol. 116, 1998
not absolutely required for protein import, it may be involved indirectly in protein translocation into peroxisomes.
DISCUSSION
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 mM 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.
(Fig. 7A). However, when the total PMF is collapsed by 10
to 20 mm CCCP, which is within the active range (Bellion
and Goodman, 1987; Imanaka et al., 1987; Nicolay et al.,
1987; Cline et al., 1992; Theg and Scott, 1993; Wendland and
Subramani, 1993), peroxisomal protein import was inhibited by approximately 40 to 50% (Fig. 7B). This suggests
that although a PMF across the peroxisomal membrane is
Energy is required to transport polypeptides through
phospholipid membranes. At least three possible sources
for that energy have been proposed: (a) energy released by
induced conformational changes caused by the initial interaction between the protein and the membrane; (b) energy released by the hydrolysis of high-energy bonds
found in ATP and other NTPs; and (c) a PMF consisting of
a transmembrane electric potential and a pH gradient
(Pugsley, 1989). Different protein import systems use each
of these sources to varying degrees. We have addressed
only the latter two in this report. Our in vitro assay for
protein import into peroxisomes allows us to biochemically
manipulate the exact reaction conditions. For most experiments, we first removed any endogenous NTPs from the
GLO translation products and then added back different
forms of energy to examine their effects on protein
translocation.
We and others have shown that peroxisomal protein
import is an ATP-dependent process (Imanaka et al., 1987;
Soto et al., 1993; Horng et al., 1995; Brickner et al., 1997),
but it is not yet known where in the targeting and translocation pathway the energy is required or how the energy
is used. One possibility is that ATP is required by cytosolic
chaperones or other cytosolic factors to ensure proper targeting of the protein to peroxisomes. In fact, the cytoplasmic ATPase PXAAA1 from humans apparently stabilizes a
soluble receptor required for peroxisomal protein import
(Yahraus et al., 1996). The Mg21 salt of ATP has been shown
to be specifically required by some chaperones for optimal
function (Miernyk, 1997). The cation specificity experiments
also showed that Mg21 is the preferred salt for protein
import into peroxisomes (Fig. 2). However, Mg21 likely has
multiple functions in this pathway. For instance, a yeast
peroxisomal, membrane-associated, proton-translocating
ATPase is also Mg21 dependent (Douma et al., 1987).
A second possible role for NTPs during peroxisomal
protein import is suggested by the nucleotide binding sites
identified on several peroxisome-associated proteins (Verheyden et al., 1992; Swartzman et al., 1996) as well as on
two components of the chloroplast translocation machinery
(Kessler et al., 1994). When isolated glyoxysomes were
preincubated with ATP and an excess of a nonhydrolyzable
NTP analog, subsequent GLO import was significantly decreased (Figs. 5 and 6), suggesting that nucleotide binding
to an unidentified factor may be important for peroxisomal
protein transport.
Both ATP and GTP were able to support GLO import in
our in vitro assay system (Figs. 1, 4–6). GTP-binding proteins are known to be involved in several other transport
pathways (Balch, 1990; Pfeffer, 1992; Kessler et al., 1994)
and three small GTP-binding proteins have been identified
in rat peroxisomal membranes (Verheyden et al., 1992).
Thus, it is possible that GTP has a direct role in peroxisomal protein import. Although Wendland and Subramani
(1993) did not observe a similar involvement of GTP-
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Energetics of Peroxisomal Protein Import
hydrolyzing proteins when they examined peroxisomal
protein import in permeabilized mammalian cells, they
used only 100 mm GTP-g-S in the presence of 1 mm ATP
and an ATP-regenerating system. The concentration of the
GTP analog may have been too low to observe a clear effect
on the localization of the microinjected peroxisomal
protein.
It is possible that ATP and GTP are both necessary for
protein import, but at distinct energy-requiring steps.
When a nonhydrolyzable analog of GTP was added to
import reactions in the presence of 5 mm ATP, the level of
GLO import was lower than in reactions containing ATP
only, but higher than the import seen with GTP-g-S alone
(Fig. 6). This reduction in the amount of GLO imported in
an ATP-dependent manner indicates that there is either a
common NTP-binding site for which ATP and GTP compete, or that the addition of GTP-g-S inhibits a separate,
GTP-requiring import step. At this time we are unable to
distinguish between these two options. It is likely that ATP
(and possibly GTP as well) has multiple roles and acts at
several different steps along the import pathway. This will
make it more complicated to determine exactly how each
nucleotide is involved in peroxisomal protein import.
Some protein transport systems also require a PMF for
maximal efficiency of translocation (Pfanner and Neupert,
1986; Yamane et al., 1987; Wong and Buckley, 1989; Cline et
al., 1992; Theg and Scott, 1993), but it is not clear whether
a PMF is present across the peroxisomal membrane. Our
results, using a broad range of ionophores, suggest that a
PMF is involved in peroxisomal protein transport, although it is not absolutely required (Fig. 7). Each of the
ionophores tested consistently reduced the amount of GLO
imported to approximately 50 to 60% of control import (in
the absence of ionophores), indicating that these ionophores inhibited protein import but did not abolish the
process. It appears that this inhibition is not caused by
decreased efficiency of import; GLO import in the presence
of ionophores does not recover even after longer reaction
times (data not shown). It may be that a PMF-requiring
factor is depleted in the presence of ionophores such that
translocation into the matrix is compromised. Alternatively, the pH of the peroxisome matrix may be important
for optimal protein import; addition of ionophores (except
valinomycin) would make it difficult to maintain a constant pH in the matrix, resulting in lower levels of peroxisomal protein import. A proton-ATPase on the peroxisome membrane may be responsible for generating and
maintaining the acidic matrix of yeast peroxisomes
(Douma et al., 1987; Nicolay et al., 1987; del Valle et al.,
1988; Waterham et al., 1990); however, neither an ATPase
nor an acidic matrix has yet been described for plant
peroxisomes.
There are two additional factors that must be considered.
First, it is important to note that low concentrations of
nigericin or valinomycin did not inhibit GLO import in
vitro (Fig. 7A). There was slightly greater inhibition of
import when nigericin and valinomycin were added together, but the results were not strictly additive (data not
315
shown). Nicolay et al. (1987) found that 1 to 2 mm nigericin
and valinomycin together was sufficient to dissipate the
pH gradient across yeast peroxisomal membranes. GLO
import into plant peroxisomes was clearly inhibited by 5 to
10 mm nigericin (Fig. 7A). When used at 10 mm, neither
valinomycin nor nigericin inhibited in vitro protein transport into rat liver peroxisomes, although no quantitation of
these results was presented (Imanaka et al., 1987). In most
systems, nigericin and valinomycin are expected to be active at very low concentrations, i.e. 1 mm or less (Pfanner
and Neupert, 1986; Cline et al., 1992; Theg and Scott, 1993).
Concentrations of nigericin or valinomycin greater than 10
mm may have nonspecific or surfactant effects (Reed, 1979).
Second, there may be some differences in the ways in
which peroxisomes from different organisms respond to
ionophores. When the total PMF was collapsed by 10 mm
CCCP, protein import into plant peroxisomes was inhibited (Fig. 7B). Wendland and Subramani (1993) found that
10 mm CCCP does not inhibit import into peroxisomes in
semipermeabilized mammalian cells; Imanaka et al. (1987)
observed no inhibition of in vitro protein transport into rat
liver peroxisomes in the presence of 10 mm CCCP. Neither
group presented any quantitation of these results. CCCP
may have more effect on yeast peroxisomes, although it is
difficult to know for sure because higher concentrations of
CCCP have been used. The assembly of alcohol oxidase is
prevented by 25 mm CCCP, leading the authors to conclude
that a PMF is required for the import and maturation of
this yeast protein (Bellion and Goodman, 1987). Using a 31P
NMR assay, Nicolay et al. (1987) found that the pH gradient across yeast peroxisomal membranes is destroyed by 2
mm CCCP.
Thus, it may be that a single component of the PMF alone
does not affect peroxisomal protein import in plants; neither nigericin nor valinomycin at low (active) concentrations inhibited GLO import (Fig. 7A). However, even 10 mm
CCCP showed maximal inhibition of GLO import (Fig. 7B),
suggesting that collapsing the total PMF decreases protein
import into peroxisomes in vitro. It seems unlikely that the
PMF is directly providing necessary energy for the translocation event. A secondary role for a PMF, such as in
maintaining a pH gradient across the membrane, may be
responsible for the consistent level of inhibition we observed in the presence of each ionophore. It is not possible
to conclude anything definitive based solely on results
provided by experiments with a single ionophore at a
single concentration. Variables such as the size of the compartment and the magnitude of the pH gradient across the
membrane may also influence the behavior of individual
ionophores (Reed, 1979). We have tested the effects on
peroxisomal protein import of a wide range of ionophores
at many concentrations, and we conclude that a PMF appears to play a role in peroxisomal protein import, perhaps
through an indirect effect, but that the energy of a PMF is
not absolutely required for protein translocation.
In a continuing effort to understand the cellular requirements and mechanisms of higher plant peroxisomal protein import, we have extensively characterized the energy
requirements for GLO import into glyoxysomes in vitro.
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316
Brickner and Olsen
We have firmly established that energy from NTPs is required during protein translocation in peroxisomes. However, future studies are needed to define exactly where and
how this energy is being used. Detailed investigations may
lead to mechanistic models that include energy-requiring
cytosolic factors and/or peroxisome-specific factors that
are necessary to translocate peroxisomal proteins through
the lipid bilayer.
ACKNOWLEDGMENTS
We thank Jason Brickner, Wendy Crookes, Yan Lin, and Aaron
Liepman for many helpful discussions. Olivia Bottum and Jessica
McHie provided excellent technical assistance. Drs. Charles Yocum and Eran Pichersky contributed useful advice and comments
during the preparation of the manuscript.
Received June 18, 1997; accepted October 8, 1997.
Copyright Clearance Center: 0032–0889/98/116/0309/09.
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