(Received for publication, April 4, 1994; and in revised form, September 13, 1994)
From the
According to current concepts, new peroxisomes are formed by division of pre-existing peroxisomes or by budding from a peroxisomal reticulum. Recent cytochemical and biochemical data indicate that protein content in peroxisomes are heterogenous and that import of newly synthesized proteins may be restricted to certain protein import-competent peroxisomal subcompartments (Yamamoto, K., and Fahimi, H. D.(1987) J. Cell Biol. 105, 713-722; Heinemann, P., and Just, W. W.(1992) FEBS Lett. 300, 179-182; Lüers, G., Hashimoto, T., Fahimi, H. D., and Völkl, A.(1993) J. Cell Biol. 121, 1271-1280).
We have observed that substantial amounts of
peroxisomal proteins are found together with ``microsomes''
(100,000 g pellet) after subcellular fractionation of
rat liver homogenates. In this study we have investigated the origin of
these peroxisomal proteins by modified gradient centrifugation
procedures in Nycodenz and by analysis of enzyme activity
distributions, Western blotting, and immunoelectron microscopy. It is
concluded that much of this material is confined to novel populations
of ``peroxisomes.'' Immunocytochemistry on gradient fractions
showed that some vesicles were enriched in acyl-CoA oxidase and
peroxisomal multifunctional enzyme (``catalase-negative'')
whereas others were enriched in catalase and thiolase (``acyl-CoA
oxidase-negative''). Double immunolabeling experiments verified
the strong heterogeneity in the protein contents of these vesicles and
also identified peroxisomes varying in size from about 0.5 µm
(``normal peroxisomes'') to extremely small vesicles of less
than 100 nm in diameter. The possibility that these vesicles may be
related to different subcompartments of a larger peroxisomal structure
involved in protein import and biogenesis will be discussed.
Peroxisomes are nearly ubiquitous organelles, generally
spherical in shape with a finely granular matrix surrounded by a single
membrane. The abundance, size, and appearance in electron microscopy of
peroxisomes vary considerably. Peroxisomes contain enzymes which
produce and degrade hydrogen peroxide, and it is clear that fatty acid
degradation is a common function of peroxisomes in eukaryotic cells
(for reviews, see (1) and (2) ). Earlier studies on
the incorporation and turnover of peroxisomal proteins suggested that
peroxisomes form a homogenous population without any signs of
maturation and with an apparently random degradation of the
organelle(3, 4) . Subsequent studies showed that
peroxisomal proteins are synthesized on unbound ribosomes and appear in
the cytosol before import into pre-existing
organelles(4, 5, 6, 7) . These data,
together with cytochemical studies(8) , showed that the
biogenesis of peroxisomes is independent of ER. ()The
current view implies that peroxisomes are formed by division of
preexisting organelles or by budding from a peroxisomal
reticulum(9, 10) . The finding that proliferation of
peroxisomes results in a heterogenous induction of peroxisomal proteins (11) may offer a useful tool in the exploration of the
mechanisms of peroxisome biogenesis. Several reports have now
demonstrated profound heterogeneity under proliferative conditions. The
first biochemical indications were obtained by analytical differential
centrifugation demonstrating that clofibrate treatment induced a
polydispersity of peroxisomes in rat liver(12) . Similar
polydispersities in rat liver occur after thyroxine
treatment(13) , ischemia reperfusion(14) , and cold
exposure(15) . A catalase-negative subpopulation of
peroxisomes, which was induced by clofibrate treatment, was identified
in mouse liver by subcellular fractionation (16) . In addition,
cytochemical studies have indicated heterogenous labeling of
peroxisomal proteins(17, 18, 19) .
Recent studies on inherited peroxisomal disorders and peroxisome assembly mutants have added much new information on the structure, function, and biogenesis of peroxisomes. The first ultrastructural observations on Zellweger syndrome indicated that the defect may be due to a total lack of peroxisomes(20) . However, subsequent studies utilizing immunocytochemistry demonstrated that peroxisomal membranes are present in cells of Zellweger patients(21, 22) , and subcellular fractionations of fibroblasts from Zellweger patients demonstrated that peroxisomal proteins are particulate to varying extents(23, 24) . Some fibroblast cell lines from Zellweger patients were able to import thiolase into peroxisomes (25) but failed to import proteins containing the carboxyl-terminal-SKL targeting signal(26) . Thus, studies on Zellweger patients have provided evidence of at least two, possibly three, distinct pathways for import of peroxisomal matrix proteins. Immunocytochemical studies on the heterogeneity of peroxisomes, and the characterization of peroxisome assembly mutants from various yeasts have shed light on both functional and developmental aspects of peroxisomes(27, 28, 29, 30, 31) . Taken together these observations now call for a revision of present peroxisome biogenesis models. A review presenting current concepts on the ultrastructural basis of the biogenesis of peroxisomes was recently published(32) .
In our earlier unpublished experiments on subcellular fractionations of liver homogenates from di(2-ethylhexyl)phthalate (DEHP)-treated rats, we found substantial amounts of peroxisomal proteins recovered in the microsomal fraction. In the present study we have further fractionated microsomal fractions by modified gradient centrifugations in Nycodenz. The enzyme activity distributions of the gradients were analyzed by Western blotting and fractions enriched in peroxisomal proteins were analyzed by immunoelectron microscopy. We conclude that with the present protocol we can separate several classes of ``peroxisomes'' from livers of DEHP-treated rats (that can be distinguished by different protein contents and sedimentation properties which are interpreted to represent peroxisomal subcompartments, probably formed during homogenization of the tissue), rather than populations of peroxisomes. These subcompartments are discussed in relation to peroxisome structure and biogenesis.
In some experiments linear Nycodenz gradients
with density range of 1.15-1.27 g/ml were prepared and
centrifuged for 1, 20, and 29-32 h at 60 000 g to compare the enzyme distributions after rate sedimentation and
equilibrium density centrifugations.
Antibodies against peroxisomal acyl-CoA oxidase (anti-Aox) and thiolase (anti-thiolase) were kindly provided by Dr. W. W. Just. Affinity-purified antibodies against peroxisomal MFE (anti-MFE) were kindly provided by Dr. J. K. Hiltunen.
Catalase was
purified from livers of DEHP-treated rats. Briefly, after
solubilization and centrifugation (at 200 000 g for 45
min in a Beckman TL-100 table top ultracentrifuge) of isolated
peroxisomes, the matrix fraction was applied to chromatography on a
DEAE column. The bound activity was eluted with a linear NaCl gradient,
and the fractions containing activity were pooled and precipitated by
35% (NH
)
SO
. The pellet was
dissolved in 50 mM Tris buffer containing 200 mM NaCl
and subjected to size exclusion chromatography in Sephacryl S-300
(Pharmacia) at a flow rate of 0.8 ml/min. The fractions containing
activity were precipitated with 65%
(NH
)
SO
and applied to a MEMSEP 1010
(DEAE cartridge, Millipore). Catalase activity was eluted with a linear
NaCl gradient (0-0.5 M) in 50 mM Tris-HCl (pH
7.4). One rabbit (of the Loop strain) was immunized intramuscularly
with 330 µg of purified catalase emulsified with Freund's
complete adjuvant, followed by three booster injections of 165 µg
of catalase protein emulsified with Freund's incomplete adjuvant.
The rabbit was bled from the ear vein at 2-week intervals after the
third booster injection, and sera were prepared. This antiserum was
monospecific for catalase at dilutions up to 1:500 000 on Western blot. (
)
Figure 1:
Subcellular fractionation of a LM
fraction in Nycodenz. A LM fraction was prepared from livers of
DEHP-treated rats and subsequently layered on top of a linear Nycodenz
density gradient ranging from 25 to 50% Nycodenz. The gradient was
centrifuged at 60,000 g for 35 min, and the gradient
was fractionated from the bottom (left to right). The gradient was analyzed for catalase and Aox
activities, and aliquots of the fractions were analyzed by Western
blotting after SDS-PAGE. The blots were probed with primary antibodies
against catalase, Aox, PMP70, and peroxisomal
thiolase.
Figure 2: Immunoelectron microscopy on low density peroxisomes isolated by centrifugation of a LM fraction in Nycodenz. The low density fraction containing highest Aox activity was prepared for immunoelectron microscopy as described under ``Methods.'' Double labeling experiments were carried out with anti-catalase (5-nm gold particles) and anti-MFE (10-nm gold particles, a and b), anti-Aox (10-nm gold particles, c and d), and anti-PMP70 (10-nm gold particles, e-g). Bar = 0.2 µm.
Figure 3:
Subcellular fractionation of a microsomal
fraction in Nycodenz. Microsomal fractions were prepared from livers of
rats treated with DEHP, layered on top of linear Nycodenz gradients,
and centrifuged at 60,000 g for 60 min. Fractions were
collected from the bottom of the tubes (from left to right) and analyzed for enzyme activities (upper
panels). It should be noted that the amount of mitochondria found
in the microsomal fractions is very low and does not contribute
significantly to the distributions of protein and 3-hydroxyacyl-CoA
dehydrogenase activity in the gradients. Gradient fractions (10 µl
of each) were electrophoresed by SDS-PAGE in 10% acrylamide gels. The
separated proteins were transferred to nitrocellulose membranes and
probed with the indicated antibodies. The blots were visualized by a
chemiluminescence method (lower
panels).
Western blot analysis of the same gradient fractions (Fig. 3, lower panel) verified the enzymatic data for catalase and 3-hydroxyacyl-CoA dehydrogenase (MFE). In addition, Western blotting showed that peroxisomal thiolase distributed similarly to catalase and that substantial amounts of PMP70 were found in fractions 15-21, indicating the presence of peroxisomal membranes in these fractions. The very close association of most of the Aox and MFE found in the microsomal fraction with microsomes after gradient centrifugation demonstrates that these proteins are not associated with peroxisomes of normal size and/or density. From these experiments it was not possible to rule out the possibility that Aox and MFE were sticking to microsomes. However, in parallel experiments where microsomal fractions were centrifuged in Percoll gradients, a clear separation of Aox activity from microsomes was obtained (data not shown).
The results shown are typical of more than 15 fractionation experiments on enzyme activity distributions and at least 5 experiments by Western blot analysis.
Figure 4: Immunoelectron microscopy on high density peroxisomes isolated by centrifugation of microsomal fractions in Nycodenz. Gradient fractions corresponding to the high density peak of peroxisomes obtained after centrifugation of microsomal fractions in Nycodenz were prepared and incubated with antibodies and colloidal gold as described under ``Methods.'' a, incubated with anti-catalase; b, incubated with anti-peroxisomal MFE; c, incubated with anti-PMP70. The peroxisomes found at high densities were generally 0.1-0.5 µm in diameter. Bar = 0.2 µm.
Figure 5: Immunoelectron microscopy on low density gradient fractions after centrifugation of microsomal fractions in Nycodenz. Microsomal fractions were centrifuged in Nycodenz gradients, and fractions corresponding to the low density peak of Aox (also containing catalase) were prepared and incubated with antibodies and colloidal gold as described under ``Methods.'' Labeled for: a and b, catalase (10 nm gold particles) and MFE (5 nm gold particles), showing a MFE-negative vesicle labeled for catalase (a) and a double-labeled vesicle positive for MFE (arrows, b); c and d, MFE (10-nm gold particles) and thiolase (5-nm gold particles). MFE-positive vesicles lacking thiolase (c) and vesicles labeled for both proteins (thiolase at arrows, d); e and f, catalase (10-nm gold particles) and PMP70 (5-nm gold particles), catalase-positive particles also labeled for PMP70 (arrows, e) and membrane material labeled for PMP70 apparently lacking catalase (f); g and h, catalase (10-nm gold particles) and thiolase (5-nm gold particles), catalase-positive particles lacking thiolase (g) and vesicles strongly labeled for thiolase (arrows, h) containing catalase. The sizes of the labeled vesicles are generally below 200 nm. Bar = 0.1 µm.
The immunolabeling experiments on the low density fractions clearly demonstrated that the peroxisomal proteins found at low density during gradient centrifugation are at least partially confined to membrane surrounded vesicles, rather than becoming released proteins in soluble form or sticking to other membranes such as microsomes. The double labeling experiments also indicated the existence of profound heterogeneities in the contents of peroxisomal enzymes in these vesicles.
Figure 6: Comparison of the sedimentation of catalase and Aox after centrifugation of microsomal fractions for 1 and 20 h in Nycodenz gradients. Liver microsomal fractions were prepared from DEHP-treated rats and centrifuged in linear Nycodenz gradients as described under ``Methods.'' The microsomal fraction was divided into three aliquots which were layered on top of Nycodenz gradients and centrifuged for 1, 20, and 29 h (not shown), respectively. The gradients were fractionated from the bottom (left to right) and analyzed for catalase and Aox activities, protein, and density. a, centrifugation for 1 h; b, centrifugation for 20 h.
Aox was also distributed into two peaks after 1-h centrifugation, one peak at about 1.22 g/ml and the other peak at about 1.15 g/ml, clearly different from catalase. After 20 and 29 h of centrifugation, Aox still tended to band in two peaks, one at about 1.22 g/ml and the other still at about 1.15 g/ml. However, it is evident that a larger part of the Aox activity also sedimented to a density of 1.25 g/ml or more, clearly higher than catalase.
These experiments imply that there exists differences also in the equilibrium densities among the vesicles containing catalase and Aox.
Isolation and characterization of peroxisomes have been hampered by the facts that peroxisomes have densities in gradient media similar to, or only slightly different from, the densities of other organelles and because peroxisomal protein normally constitutes only 2% (in rat liver) or less of the cellular protein. It is also well established that peroxisomes are leaky and that the leakiness is selective. Catalase and thiolase are very easily released, whereas other proteins, such as Aox and the MFE, are much less prone to be released(42) . Other peroxisomal proteins may be expected to cover the range. This leakiness appears to be time-dependent and dependent on the treatment of peroxisomes. It has also been implied that peroxisomes are very sensitive to hydrostatic pressure, since enzyme distributions in gradients often show activity of peroxisomal proteins throughout the gradients. However, in the light of the results presented here, the peroxisomal proteins found in the low density fractions are at least partially present in vesicles.
The most likely interpretation of our results is that the different peroxisomal subcompartments described here are formed during homogenization of a peroxisome reticulum. Fig. 7illustrates how the different peroxisomal populations, found during subcellular fractionation of rat liver homogenates, may form by vesiculation. Besides normal peroxisomes (1), the peroxisomes found at a density of about 1.19 g/ml (corresponding to the high density peaks after centrifugation of microsomes) may be formed by the pinching off of buds that are not yet complete (2). The very small vesicles with heterogenous protein contents may be formed by vesiculation of the ``stalks'' connecting the buds to the ``body'' of the reticulum (3, 4, 5). The heterogeneity of these vesicles could be explained by a nonrandom distribution of the receptors involved in binding and import of peroxisomal proteins. Such a heterogeneity could also explain why we are able to isolate different populations of peroxisomes enriched in proteins containing different PTS.
Figure 7: Hypothetical model for the formation of various populations of peroxisomes during subcellular fractionation of rat liver. If peroxisomes are formed by budding from a peroxisomal reticulum, fragmentation of such a reticulum by homogenization could explain the observed heterogeneity of vesicles containing peroxisomal proteins. Normal peroxisomes (1) are formed by budding and correspond to the bulk of peroxisomes found at densities around 1.24 g/ml. Fragmentation of ``buds'' (at the stalks) may thus correspond to ``immature'' peroxisomes with a complete set of peroxisomal proteins sedimenting to about 1.19 g/ml (2). Fragmentation of the stalks can be expected to result in the formation of ``microperoxisomes'' (3, Aox- and MFE-enriched vesicles; 4, catalase-enriched vesicles; and 5 thiolase-enriched vesicles). The heterogeneity could be explained by uneven distribution of receptors for import of peroxisomal proteins. It is possible that the ``body'' of the reticulum forms another population (?) of vesicles that is not yet identified due to lack of an appropriate marker.
Recent information concerning
peroxisome structure and protein content in Zellweger syndrome has
demonstrated that peroxisomal particles may have a selective set of
proteins. The presence of peroxisomal ghosts containing peroxisomal
membrane proteins(21, 22) , and the finding of a low
density particle (w-particle) in fibroblasts of Zellweger patients
which contained catalase(24) , also demonstrated a
heterogeneity in peroxisomal protein content. Suzuki et al.(23) showed by indirect immunofluorescence staining and
subcellular fractionation that some catalase and Aox was associated
with organelles in some fibroblasts from Zellweger patients.
Interestingly, substantial amounts of 3-ketoacyl-CoA thiolase and PMP70
were detected in organelles in all cells. These data suggest that the
transport and processing of peroxisomal proteins carrying different
PTS's are different, which is compatible with the present
knowledge on sequence requirements for import of these proteins into
peroxisomes. Several peroxisomal proteins contain a conserved
COOH-terminal -Ser-Lys-Leu sequence, or acceptable variations of this
sequence, that directs proteins to microbodies in mammalian cells,
yeasts, plants, insects, and
trypanosomes(54, 55, 56, 57, 58, 59, 60, 61) .
Import of proteins containing this conserved tripeptide is probably
mediated by a common receptor. However, import of peroxisomal thiolase
appears to be dependent on a cleavable NH-terminal
presequence(59, 62, 63) . The targeting
sequence for catalase is not clear at present. It appears that the
internal -Ser-His-Leu-sequence may not target catalase to peroxisomes,
as it was demonstrated that the addition of one or two amino acids
COOH-terminal of the -SKL PTS abolishes import into
peroxisomes(64) . This implies that catalase may follow
another, still unknown, mechanism for targeting and import.
Fahimi et al.(32) suggested that synthesis and incorporation
of membranous material, containing PMP70, is an early event, resulting
in the growth of the membrane followed by import of newly synthesized
proteins. Our results are compatible with such a model favoring
synthesis of membrane material as an early event which is followed by
incorporation of highly expressed -oxidation enzymes (Aox, MFE,
and thiolase). This is reflected by the high content of Aox and MFE in
the low density compartment after DEHP treatment. However, neither this
model nor our present results can fully explain the detailed mechanism
for the heterogenous distribution of peroxisomal proteins in different
peroxisomes.
Strong support for this model is also
obtained from studies in yeasts where a functional heterogeneity among
microbodies has been suggested to exist(27, 28) .
These studies demonstrated that addition of methanol to Candida
boidinii cells grown on oleic acid resulted in incorporation of
newly synthesized proteins into smaller, presumably
``immature'' peroxisomes. This heterogeneity was only
transient, and continuous cultures grown on a mixture of oleic acid and
methanol at steady-state conditions showed that both the enzymes of the
-oxidation pathway and methanol metabolism were found in one and
the same compartment(28) . This transient heterogeneity is
likely to reflect an incompetence of mature peroxisomes to incorporate
newly synthesized proteins. This idea is further supported by the
recent pulse-chase experiments on in vitro import which showed
that Aox is imported into a compartment of intermediate density (pulse)
and subsequently chased into a high density compartment (normal
peroxisomes)(67) . During our present investigation,
Lüers et al.(41) reported on a
modified scheme of subcellular fractionation of regenerating livers
which resulted in the separation of two apparently different
populations of peroxisomes. They found that
[
S]methionine injected into rats was first
incorporated into a ``light peroxisomal'' fraction
(5-30 min after injection) and that the label appeared in the
``heavy peroxisomal'' fraction at 90 min.
The formation of a peroxisomal reticulum distinguishes peroxisomes from the other intracellular membrane systems of mitochondria, chloroplasts, and lysosomes. Mitochondria are generally believed to represent an ancient endosymbiont, probably after ``invasion'' of a bacteria. ER and lysosomal proteins are synthesized by co-translational insertion of the proteins into ER followed by glycosylation. ER resident proteins are apparently retrieved in the ER by a receptor-mediated process involving a COOH-terminal tetrapeptide (KDEL or HXEL) (see (68) for review). Lysosomal proteins are transported to the Golgi apparatus where the lysosomes are formed by budding of vesicles containing lysosomal proteins which are then transported to the acidic compartment. Peroxisomal proteins may be transported from the site of import (at the peroxisome-forming sheet) by ``bulk flow'' (similar to protein transport in the rough ER) to the buds. Experiments aimed at functionally characterizing the different peroxisomal subcompartments, which are under way in our laboratory, may clarify the origin and functions of these vesicles. It still remains to biochemically identify the peroxisome-forming sheet during subcellular fractionation of rat liver homogenates.