(Received for publication, June 28, 1995; and in revised form, November 2, 1995)
From the
Biosynthesis and intracellular transport of 70-kDa peroxisomal
membrane protein (PMP70) has been studied in rat hepatoma, H-4-II-E
cells. Pulse-chase analysis showed that a newly synthesized S-PMP70 first appeared in the cytosolic fraction and was
then transported into the peroxisomal fraction. The half-life of
S-PMP70 in the cytosolic fraction was approximately 3 min.
Integration of
S-PMP70 into membranes occurred in the
peroxisomal fraction and was completed within 30 min. No proteolytic
processing of
S-PMP70 was observed. An in vitro import system was reconstituted to characterize the insertion
mechanism of PMP70 into peroxisomes. Peroxisomes isolated from rat
liver were incubated at 26 °C with
[
S]methionine-labeled in vitro translation products of PMP70 mRNA in the presence of the
cytosolic fraction. The peroxisomes were reisolated and insertion of
S-PMP70 into the membrane was analyzed using a
Na
CO
procedure. The binding and insertion of
S-PMP70 were dependent on temperature and incubation time
and was specific for peroxisomes. Pretreatment of peroxisomes with
trypsin and chymotrypsin almost abolished the binding and insertion of
S-PMP70. The translation products contained several
truncated
S-PMP70s. The NH
terminally
truncated
S-PMP70s, with a molecular mass greater than 50
kDa, bound to and inserted into peroxisomal membranes, whereas
truncated
S-PMP70s smaller than 45 kDa did not. These
results suggest that PMP70 is post-translationally transported to
peroxisomes without processing and inserted into peroxisomal membranes
by a specific mechanism in which a proteinaceous receptor and a certain
internal sequence of PMP70 are involved.
Peroxisomes are organelles bounded by a single membrane which
are present in almost all eukaryotic cells. The peroxisomes are
involved in a variety of metabolic processes including peroxide-based
respiration, oxidative degradation of purines and fatty acids, and
synthesis of plasmalogen and bile acids(1, 2) . It has
recently been suggested that peroxisomes are formed by
post-translational import of newly synthesized proteins into
pre-existing peroxisomes, which then divide (3, 4, 5) . In rat liver, it is known that
matrix proteins in peroxisomes are all synthesized on free polysomes in
the cytosol. Post-translational import of these proteins into
peroxisomes has been shown by in vivo pulse-chase (6, 7) and also by in vitro import
experiments(8, 9, 10, 11, 12) .
We developed an in vitro system based on the import of
radiolabeled acyl-CoA oxidase (AOx) ()into purified rat
liver peroxisomes and showed that ATP hydrolysis was required for the
translocation of AOx through peroxisomal membranes(9) . At
least two types of peroxisomal targeting sequences (PTSs) have been
found. PTS1 consists of the sequence Ser-Lys-Leu (or closely related)
at the carboxyl terminus (10, 13) and PTS2, which in
3-ketoacyl-CoA thiolase is found in the 11 amino acids of the
amino-terminal leader sequence(14, 15) .
The membranes of rat liver peroxisomes contain several integral membrane proteins not found in other organelles. Polypeptides of 69/70 and 22 kDa have been identified as major components and polypeptides of 57, 53, 35/36, and 26/27 kDa have been identified as minor components of peroxisomal membranes(16, 17, 18, 19) . The 70-kDa peroxisomal membrane protein (PMP70) is markedly induced by administration of hypolipidemic agents and parallels peroxisome proliferation(17, 18) . We cloned and sequenced PMP70 and found that PMP70 belonged to a superfamily called an ATP-binding cassette protein, conferring multidrug resistance to tumor cells(20) . Recently PMP70 was suggested to be essential for peroxisome formation(21) . PMP22 was also cloned and sequenced and it was suggested that the topology of PMP22 was similar to those of Mpv 17 and certain transmitter gated ion channels, although the function of PMP22 has not yet been described(22) . Recently peroxisome assembly factor 1, a peroxisomal integral membrane protein with molecular mass of 35 kDa was also cloned and sequenced(23) .
A number of studies have been carried out to further investigate the biogenesis of peroxisomal membrane proteins (PMPs). We, and other laboratories, have shown that PMP70, -26, and -22 were synthesized on free polysomes(24, 25, 26) , suggesting that the PMPs are likely to be inserted post-translationally into peroxisomal membranes. However, the intracellular transport of any PMP has not yet been investigated. The occurrence of this event should be confirmed before establishing an in vitro import system.
As a preliminary step to understanding the molecular mechanism of assembly of PMPs, we carried out pulse-chase experiments to investigate the kinetics of intracellular transport of newly synthesized PMP70 in rat hepatoma H-4-II-E cells. From these results, we have been able to develop an in vitro import system using isolated rat liver peroxisomes and in vitro translation products of PMP70 mRNA. We obtained experimental evidence showing that PMP70 was post-translationally transported to peroxisomes and then inserted into their membranes without processing. In addition we found that the binding and insertion of PMP70 was mediated by a specific mechanism in which a proteinaceous receptor and a certain internal sequence of PMP70 may be involved.
Figure 1:
Subcellular
localization of PMP70 in H-4-II-E cells. A, H-4-II-E cells
were fractionated by differential centifugation into nuclear fraction (N), mitochondrial fraction (M), light mitochondrial
fraction (LM), and microsomal and cytosolic fraction (Mc+S). Catalase, cytochrome c oxidase (Cyt
Ox), and NADPH-cytochrome c reductase (Cyt Red)
were measured as marker enzymes of peroxisomes, mitochondria, and
microsomes, respectively. The recoveries varied between 80 and 115%.
Distribution of PMP70 was calculated from scanning densitometry of the
autoradiogram shown in B. B, immunoblot analysis of
PMP70. Membranes in each subcellular fraction were prepared by a
NaCO
procedure. Applied samples for immunoblot
analysis were derived from 50 to 200 µg of protein of each
subcellular fraction. C, a light mitochondrial fraction from
H-4-II-E was fractionated by sucrose gradient centrifugation. N-Acetyl-
-D-glucosaminidase (NAGase)
and lactate dehydrogenase (LDH) were measured as marker
enzymes of lysosomes and cytosol, respectively. The recoveries varied
between 70 and 120%. D, immunoblot analysis of PMP70.
Membranes were prepared from 100 µl of each fraction from the top (No. 1) to the bottom (No. 11) of the
gradient.
Figure 2:
Synthesis and intracellular transport of
PMP70 in H-4-II-E cells. H-4-II-E cells were pulsed for 5 min and
chased for 0-60 min. The cells at each chase period were
homogenized and PNS fractions were prepared. A, each PNS
fraction was fractionated into supernatant (post-peroxisomal
supernatant) () and particulate (
) fractions by
centrifugation at 16,000
g for 20 min. Aliquots (200
µl) of each fraction (1.0 ml) were subjected to
immunoprecipitation. B, PNS fractions prepared in a separate
chase experiment were fractionated by sucrose density centrifugation. A
portion (500 µl) of each fraction was subjected to
immunoprecipitation. C, portions of the PNS fractions obtained
in A were fractionated into soluble (S) and membrane (P) fractions by Na
CO
treatment. Both
fractions were subjected to
immunoprecipitation.
To
analyze the kinetics of S-PMP70 integration into
membranes, PNS fractions isolated after different chase periods were
separated into membrane and soluble fractions by a
Na
CO
procedure. As shown in Fig. 2C, lanes 1 and 2, immediately
after labeling the cell almost all the
S-PMP70 was
recovered in the soluble fraction, suggesting that the protein had not
been integrated into any membrane. After chase periods of 2.5-15
min, the amount of
S-PMP70 increased gradually in the
membrane fraction (lanes 3-12) and almost 100% of
S-PMP70 was recovered in the membrane fraction after 30
min (lanes 13 and 14).
Quantification of the
radioactivity was achieved by densitometric scanning of the
autoradiographs in Fig. 2. As shown in Fig. 3, newly
synthesized S-PMP70 appeared first in the cytosol, after
which it transferred to the peroxisomes with a half-time of
approximately 3 min. The time course of
S-PMP70
integration into membranes was delayed in comparison to that of
S-PMP70 association with peroxisomes. These results
suggest that PMP70 is associated post-translationally to peroxisomes
and then integrated into their membranes.
Figure 3:
Kinetics of intracellular transport of
PMP70 after pulse-chase labeling. The points represent the
radioactivities of immunoprecipitates of Fig. 2. ,
particulate fraction;
, supernatant fraction;
, membrane
fraction after Na
CO
treatment.
Figure 4: In vitro synthesis of PMP 70. PMP70 was synthesized from a cDNA by coupling in vitro transcription and translation (lane 1). The in vitro translation products were immunoprecipitated with preimmune serum (lane 2), anti-PMP70 (lane 3), or anti-COOH-terminal 15 amino acids of PMP70 (lane 4).
Figure 5:
In vitro import of PMP70 to rat
liver peroxisomes. The in vitro translation products of PMP70
mRNA were mixed at 0 °C with or without rat liver peroxisomes (100
or 200 µg) in a volume of 250 µl of SEH containing 1 mM ATP, 3 mM MgCl, 50 mM KCl, and 1.5
mg of cytosolic proteins as described under ``Experimental
Procedures.'' After incubation for 60 min at 26 or 0 °C, the
peroxisomes were separated into the supernatant (S) and pellet (P) by centrifugation at 14,000
g for 20 min. A, in vitro association of PMP70 to peroxisomes.
Aliquots of P and S fractions were analyzed by
SDS-PAGE and fluorography. T, total translation products
corresponding to 50% of the input radioactivity. B,
peroxisomal pellets after in vitro import experiment were
fractionated in soluble (Sol) and membrane (Mb)
fractions by Na
CO
treatment. Aliquots of both
fractions were analyzed by SDS-PAGE and
fluorography.
Figure 6:
Time dependent insertion of PMP70. The
insertion assay was carried out with 200 µg of peroxisomal protein
and peroxisomal membranes were prepared by NaCO
treatment. Radioactivity is expressed in arbitrary units as the
area of each peak obtained by densitometric scanning of 70-kDa protein
band.
Figure 7: Organelle specificity of the import of PMP70. The import assay was carried out with equal amounts of peroxisomal and mitochondrial protein. A, association of PMP70 with peroxisomes or mitochondria. T, total translation products corresponding to 50% of the input radioactivity. B, insertion of PMP70 into peroxisomal membranes. S, supernatant; P, pellet; Sol, soluble; Mb, membrane.
Figure 8:
Effect of mild trypsin and chymotrypsin
pretreatment on the ability of peroxisomes to import PMP70. Peroxisomes
(600 µg) were treated with trypsin and chymotrypsin (6 µg each)
in 100 µl of 0.25 M SVE containing 5 mM Hepes/KOH, pH 7.4, at 0 °C for 10 min. After incubation,
protease inhibitors (30 µg of soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin,
chymostatin, antipain, and pepstatin A) were added to the reaction
mixture. The samples were diluted 10-fold with the above solution and
centrifuged at 16,000 g for 20 min and the peroxisomal
pellets were resuspended in 0.25 M SEH. A,
peroxisomal proteins stained with Coomassie Brilliant Blue (CBB). B, association of PMP70 to peroxisomes. Import
assay was carried out with 150 µg of peroxisomal protein. T, total translation products corresponding to 50% of the
input radioactivity. S, supernatant; P,
pellet.
Figure 9: Effect of NEM on the import of PMP70. Isolated peroxisomes, cytosol, and translation products of PMP70 mRNA were treated with 1 mM NEM for 10 min at 26 °C. After incubation excess NEM was blocked by the addition of 2.5 mM dithiothreitol. A, association; and B, insertion. Lanes 2 and 3, control (no treatment of NEM); lanes 4 and 5, NEM-treated peroxisomes; lanes 6 and 7, NEM-treated cytosol; lanes 8 and 9, NEM-treated translation products; lanes 10 and 11, NEM-treated peroxisomes, cytosol, and translation products. T, total translation products corresponding to 50% of the input radioactivity. C, import of AOx into peroxisomes. Import assay was carried out with control or NEM-treated peroxisomes. At the end of import assay, one of duplicated samples (+) was treated with 4 µg of proteinase K at 0 °C for 15 min. The peroxisomes were pelleted by centrifugation and analyzed by SDS-PAGE and fluorography according to the method described in (31) . HD, hydratase-dehydrogenase. T, total translation products corresponding to 20% of input radioactivity.
The
translocation of AOx, firefly luciferase, and human serum albumin with
PTS through peroxisomal membranes has been shown to require ATP
hydrolysis both in vitro(9) and in a permealized cell
system(33, 34) . As shown in Fig. 10, depletion
of ATP with apyrase did not reduce association and insertion of S-PMP70. Under this condition, the ATP content of the
import mixture was less than 5 µM, a concentration too
small to enable the translocation of AOx into peroxisomes(9) .
Treatment of the import mixture with heat inactivated enzyme also had
no effect.
Figure 10: Effect of ATP on the import of PMP70. Depletion of ATP from the import assay. Peroxisomes were incubated with translation products and cytosol which were treated either with apyrase (1.4 units/250 µl) at 26 °C for 10 min or with heat inactivate apyrase in order to deplete ATP from translation products and cytosol. A, association; B, insertion. Lanes 2 and 3, control; lanes 4 and 5, active apyrase; lanes 6 and 7, inactivate apyrase. T, total translation products corresponding to 50% of the input radioactivity. S, supernatant; P, pellet; Sol, soluble; Mb, membrane.
In light of these experiments, pulse-chase studies were carried out. It was shown that PMP70 was synthesized by free polysomes in the cytosol, and then rapidly transferred post-translationally to peroxisomal membranes. The initial cytosolic location of PMP70 was shown by the fact that the newly synthesized PMP70 was mainly in the cytosolic fraction at chase 0 min ( Fig. 2and Fig. 3). The kinetics of PMP70 transport suggest that PMP70 has a half-life in the cytosol of approximately 3 min. In previous studies of the post-translational import of peroxisomal matrix proteins, rat liver urate oxidase and hydratase-dehydrogenase have a half-life in the cytosol of <5 min, catalase has 14 min (6) and yeast Candida boidinii alcohol oxidase 20 min(38) . Thus PMP70 is rapidly transported to peroxisomes as on a similar time scale to urate oxidase and hydratase-dehydrogenase. In addition, insertion of PMP70 into membranes was delayed slightly compared to the binding of PMP70 to peroxisomes (Fig. 3), suggesting that newly synthesized PMP70 is transported to peroxisomes and then integrated into their membranes. Additional studies showed that PMP70 is synthesized at the mature size with no detectable proteolytic processing occurring upon transfer into the peroxisomal membranes. This is in agreement with previous studies which showed that the size of the translation product of PMP70 was indistinguishable from PMP70 present in peroxisomes(25, 26) .
It seems unlikely that newly
synthesized PMP70 is first transported to the endoplasmic reticulum and
then buds as small membrane vesicles targeted to peroxisomes. Since S-PMP70 in the postperoxisomal fraction at 0 min chase was
not sedimented by centrifugation at 100,000
g for 1 h,
while almost all the microsomes were recovered in the pellet (data not
shown). Furthermore, in vitro translation products of PMP70
mRNA were neither associated with nor inserted into microsomal
membranes in an in vitro import assay (data not shown).
The organelle specificity suggest that specific factor(s) on peroxisomal membranes may participate in binding and/or insertion of PMP70. The existence of a protein which mediates the binding and insertion is supported by the present finding that mild proteolytic pretreatment of the peroxisomes abolished both binding and insertion of PMP70 into the peroxisomal membranes (Fig. 8). We do not yet know which proteins(s) is responsible, since even under the mild conditions employed a number of PMPs were damaged. However, it appears unlikely that the essential protein is one of the small PMPs such as 22, 26/27, and 35/36 kDa, since these remained undamaged under conditions where PMP70 insertion was markedly reduced (data not shown).
Another important point addressed by the present study is the
topogenic sequence of PMP70. It is suggested that PMP70 contains six
putative transmembranes and the ATP-binding motif at the
carboxyl-terminal is exposed to the cytosol, although the exact
location of the amino terminus has not yet been
determined(20) . PMP70 must use a targeting signal other than
PST1 or PST2, since PMP70 has PST1- or PST2-like sequences at the COOH
or NH terminus(20) . As shown in the case of
mitochondrial outer membrane proteins(39) , the transmembrane
domains of PMP70 will be necessary to anchor it in the membranes and a
signal sequence near the stop-anchor sequence will be required for
specific association with only the peroxisomal membranes. The truncated
S-PMP70s greater than 50 kDa bound to and inserted into
peroxisomal membranes, whereas the truncated
S-PMP70s
smaller than 45 kDa did not (Fig. 5). This association of
several truncated PMP70s to peroxisomes gives a clue as to the position
of the signal sequence of PMP70. Furthermore, the efficiency of
association and insertion of truncated
S-PMP70s greater
than 50 kDa seem to be essentially the same ( Fig. 5and Fig. 7-10). These results suggest that an internal region
of PMP70, especially the region around 20-25 kDa from the
NH
-terminal is necessary for peroxisomal targeting and
insertion. This region roughly corresponds to the predicted third
transmembrane sequences. Recently McCammon et al.(40) suggested that PMP47 of the methylotrophic yeast, C. boidinii, possess a peroxisomal targeting sequence
in an internal region, which nearly corresponds to the fourth of six
putative transmembrane regions.
The insertion mechanism of PMP70 into peroxisomal membranes seems to be different from the translocation mechanism of several peroxisomal matrix proteins. In the case of the matrix protein import, ATP hydrolysis (9, 33, 34) and NEM-sensitive component(s) of peroxisomal membranes (33) are required. However, insertion of PMP70 into peroxisomal membranes did not need ATP (Fig. 10) and NEM-sensitive component(s) (Fig. 9). It remains, however, unresolved whether ATP or the NEM-sensitive factor is required for correct folding of PMP70.
Very recently Diestelkotter and Just (41) demonstrated that PMP22 was inserted into peroxisomal
membranes of liver prepared from clofibrate-treated rat in
vitro. The insertion of S-PMP22 into peroxisomes
seems to be mediated by a proteinaceous receptor, did not require ATP,
and was not inhibited by NEM treatment. The insertion of PMP70 seems to
be facilitated by a similar mechanism to PMP22. However, the
association of PMP22 to peroxisomal membranes occurred at 0 °C and
26 °C with the same efficiency, whereas the association of PMP70
did not occur at 0 °C, suggesting that binding of PMP70 and PMP22
have different features. Different types of receptor(s) or cytosolic
factor(s) may facilitate binding of PMP70 and PMP22. A detailed
comparison of both insertion mechanisms will be the subject of future
research.
In this study, we confirmed that PMP70 was post-translationally transported to and inserted into peroxisomal membranes without processing. Furthermore, we suggest that a proteinaceous receptor and a certain internal sequence of PMP70 might be involved in the binding and the insertion of PMP70 into the peroxisomal membranes. Identification of the receptor as well as the minimum topogenic sequence are now under investigation.