Biogenesis of Nonspecific Lipid Transfer Protein and Sterol
Carrier Protein x
STUDIES USING PEROXISOME ASSEMBLY-DEFECTIVE pex CELL
MUTANTS*
Hidenori
Otera
,
Maki
Nishimura
,
Kiyoko
Setoguchi
,
Takeshi
Mori§, and
Yukio
Fujiki
¶
From the
Department of Biology, Faculty of Sciences,
Kyushu University Graduate School, Fukuoka 812-8581,
§ Meiji Milk Products Co., Odawara, Kanagawa 250-0862, and
¶ CREST, Japan Science and Technology Corporation, Tokyo
170-0013, Japan
Received for publication, August 24, 2000, and in revised form, October 18, 2000
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ABSTRACT |
Nonspecific lipid transfer protein (nsLTP; also
called sterol carrier protein 2) with a molecular mass of 13 kDa is
synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal
20-amino acid extension presequence, as well as with the peroxisome
targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The
precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at
the C-terminal part and is converted to the 46-kDa form and nsLTP after
the transport to peroxisomes. We investigated which of these two
potential topogenic sequences functions in biogenesis of nsLTP and
SCPx. Morphological and biochemical analyses, making use of Chinese
hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective
pex7, as well as green fluorescent protein chimeras,
revealed that both pre-nsLTP and SCPx are imported into peroxisomes by
the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains
in the cytosol, as assessed by subcellular fractionation of the
wild-type Chinese hamster ovary cells. In an in vitro
binding assay, only mature nsLTP, but not pre-nsLTP, from the cell
lysates interacted with the Pex5p. It is likely, therefore, that
modulation of the C-terminal PTS1 by the presequence gives rise to
cytoplasmic localization of pre-nsLTP.
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INTRODUCTION |
The mammalian nonspecific lipid transfer protein
(nsLTP)1 functions in
vitro as a carrier protein in the transfer of a variety of lipids,
such as phospholipids and cholesterol (for a review, see Ref. 1). It is
identical to sterol carrier protein 2, stimulates the enzymatic
conversion of lanosterol to cholesterol during the biosynthesis of
cholesterol, and enhances the synthesis of bile acids (1). But, the
physiological role of nsLTP is not yet well defined. nsLTP is localized
in peroxisomes, as well as the cytoplasm, in several organs such as rat
liver (2, 3) and adrenal gland (1). nsLTP is synthesized as a larger
precursor, termed pre-nsLTP, with a mass of 15 kDa on free
polyribosomes (4) and then processed to its mature form of 13 kDa (4, 5), apparently after transport into peroxisomes (2, 4). Two types of
cDNA for nsLTP of rat liver were cloned (6-8), one encoding 15-kDa
pre-nsLTP consisting of 143 amino acids, a precursor form of 123-amino
acid mature nsLTP (9, 10), and the other coding for a 547-amino acid
protein with a mass of 59 kDa, termed sterol carrier protein x (SCPx)
(see Fig. 1A).
pre-nsLTP and SCPx are expressed from one gene by alternative
transcription initiation by two distinct promotors (11). The N-terminal, 20-amino acid extra sequence of pre-nsLTP is cleaved off to
form mature nsLTP in peroxisomes (4, 12). Interestingly, nsLTP was
recently shown to interact with acyl-CoA oxidase (AOx) (13), the
first-step enzyme of peroxisomal
-oxidation system, suggesting that
nsLTP may function in transfer of the substrates such as fatty acyl-CoA
derivatives to AOx. The sequence for SCPx contains the full sequence of
pre-nsLTP at the C-terminal part and is suggested to be partly
converted to the N-terminal part protein of 46 kDa and nsLTP (1, 14).
It has been demonstrated that SCPx functions as a peroxisomal
branched-chain
-ketothiolase (15-17).
The import of most peroxisomal matrix proteins is mediated by well
characterized cis-acting peroxisomal targeting signals (PTSs), C-terminal Ser-Lys-Leu (SKL) motif PTS1 (18, 19) and N-terminal
cleavable presequence PTS2 (20-22). Other types of PTS have been
postulated to exist but have not been identified yet. It is evident
that pre-nsLTP contains a cleavable N-terminal 20-amino acid
presequence resembling PTS2, as well as PTS1 tripeptide Ala-Lys-Leu (AKL), whereas SCPx possesses internally this 20-amino acid presequence of nsLTP and the C-terminal AKL. However, despite numerous biochemical findings such as those related to the lipid transfer activity, biogenesis of nsLTP and SCPx has not been well defined at the molecular
and cellular levels.
To address the underlying mechanisms by which nsLTP and SCPx are
transported into peroxisomes, we investigated biogenesis of these
proteins at morphological, as well as biochemical, levels using several
types of peroxisome biogenesis-defective cell mutants and the PTS1
receptor Pex5p. We herein report that import of nsLTP and SCPx was
affected in pex5 mutant of Chinese hamster ovary (CHO) cells
deficient in import of PTS1 and PTS2, whereas in PTS2 import-defective
pex7 mutant both proteins were imported into peroxisomes as
efficiently as in the wild-type CHO-K1. We also observed that nsLTP and
SCPx bound to Pex5p. Therefore, these findings strongly suggest that
nsLTP and SCPx are imported by the PTS1 translocation pathway. The
function of the 20-amino acid presequence in intracellular transport of
pre-nsLTP is also discussed.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
Wild-type CHO-K1 and pex mutants,
including pex2 (23), pex5 (24), and
pex7 (25) (Table I),
were cultured as described (26).
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Table I
CHO cell pex mutants used in this study and their complementing
genes
Protein import: +, normal; , impaired.
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Antibodies--
Affinity-purified rabbit anti-rat nsLTP antibody
was as described (2, 4). Rat cDNA (TM-33/34) encoding the 59-kDa
SCPx protein (6) was amplified by polymerase chain reaction using SCPx
primers, a forward 5'-CCCGGGAACTCTCCGCGCCTGCGC-3'
(underline, SmaI site) and a reverse
5'-GTCGACATCACTGGGACCCAGGCC-3' (underline, SalI site). The polymerase chain reaction product was cloned
into pGEM-T Easy vector (Promega) and cleaved with SmaI and
SalI. The SmaI-SalI fragment
encompassing an N-terminal part consisting of amino acid residues at
7-300 of 59-kDa SCPx was inserted into the
SmaI-SalI site of pUEX3 (Amersham Pharmacia
Biotech). The resultant plasmid for lacZ fusion protein was expressed
in Escherichia coli. Antiserum specific for SCPx was raised
in rabbit by conventional subcutaneous injection of
-galactosidase-SCPx fusion protein. Rabbit anti-green fluorescent
protein (GFP) antibody was purchased from CLONTECH.
Morphological Analysis--
nsLTP and SCPx in CHO cells were
visualized by indirect immunofluorescence light microscopy using rabbit
antibodies to nsLTP and SCPx, respectively, as described (27). We also
used goat anti-rat catalase antibody (28) and rabbit anti-malate
dehydrogenase antibody (29). Antigen·antibody complexes were
detected under a Carl Zeiss Axioskop FL microscope by fluorescein
isothiocyanate-labeled sheep anti-rabbit IgG antibody (Cappel), Texas
Red-labeled sheep anti-rabbit IgG antibody (Cappel), or donkey
anti-goat IgG antibody conjugated to rhodamine (Chemicon).
Subcellular Fractionation--
Subcellular fractions of rat
liver and CHO cells were prepared as described (19, 30). A high speed
supernatant, termed cytosolic, fraction was prepared by centrifuging a
post-nuclear supernatant fraction (PNS) for 40 min at 100,000 × g (30). Isopycnic ultracentrifugation on a sucrose density
gradient using PNS from CHO cells was done as described (30).
In Vitro Binding Assay--
Cell lysates were prepared from
CHO-K1 cells using 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 25 µg/ml each
of leupeptin and antipain, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM dithiothreitol as
described (30). In vitro binding assays were performed by
incubation of cell lysates with glutathione S-transferase
(GST) fused to the longer isoform of Pex5p (Pex5pL) and
GST-Pex5pL-G335E with a mutation at G335E derived from a
pex5 mutant, ZP105, as described (30). Bound proteins were
analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and immunoblot.
Expression of GFP Fusion Protein--
DNA fragment for the
N-terminal presequence of nsLTP was obtained by polymerase chain
reaction using as a template cDNA encoding pre-nsLTP (6) in pUcD2
and a set of primers, a forward primer nsLTP-XbaI,
CCTCTAGAAGAATGGGTTTTCCC, and a reverse nsLTP-BamHI, AATGGATCCCCTGCAGAGCTGGT. After digesting with XbaI and
BamHI, the resulting fragment was inserted into the
XbaI-BamHI of pUcD2HygGFP(TT) (28), termed
pUcD2Hyg·pre-GFP. pUcD2Hyg·pre-GFP-AKL was
constructed as follows: PmaCI-KpnI fragment of
pUcD2Hyg·GFP-AKL (28) was inserted into the
PmaCI-KpnI site of pUcD2Hyg·pre-GFP.
The resultant plasmids were assessed by nucleotide sequencing using a
Dye-terminator DNA sequence kit (Applied Biosystems). Cells expressing
GFP that had been grown on a cover glass were observed after cell
fixation (28) under a Carl Zeiss Axioskop FL microscope using a number 17 filter.
Other Methods--
Western blot analysis on polyvinylidene
difluoride membrane (Bio-Rad) was performed using primary antibodies
including rabbit anti-AOx antibody (26) and a second antibody, donkey
anti-rabbit IgG antibody conjugated to horseradish peroxidase (Amersham
Pharmacia Biotech). Antigen·antibody complexes were detected using
ECL Western blotting detection reagent (Amersham Pharmacia Biotech).
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RESULTS |
Anti-SCPx Antibody--
We raised antiserum against SCPx by
immunizing a rabbit with SCPx (residues at 7-300; see Fig.
1A) fused to E. coli
-galactosidase. The antibody specifically recognized 59- and 46-kDa proteins in PNS, as well as in purified peroxisomes (Fig.
1B, lanes 1 and 2). The 46-kDa protein
is most likely to be derived from 59-kDa SCPx by proteolytic conversion
(1, 14). Using anti-nsLTP antibody, 13-kDa, as well as 59-kDa, proteins
were detected in PNS and peroxisomes (Fig. 1B, lanes
3 and 4), consistent with our earlier observation (2,
4). In cytosolic fraction ~15 kDa of protein reacted with this
antibody, in addition to 13- and 59-kDa polypeptides (lane
5), both presumably from broken peroxisomes during homogenization
of liver (2). We interpreted these results to mean that nsLTP and
59-kDa SCPx cross-reactive to anti-nsLTP antibody are localized in
peroxisomes, and pre-nsLTP is present in the cytosol. It is more likely
that SCPx is partly processed to produce nsLTP and an N-terminal 46-kDa
thiolase protein of SCPx (1, 14). These data demonstrate that both
antibodies are specific.

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Fig. 1.
Specificity of antibodies raised against
nsLTP and SCPx. A, schematic view of SCPx and
pre-nsLTP. Filled and hatched bars indicate the
20-amino acid presequence and mature protein of nsLTP, respectively.
Intraperoxisomal cleavage site of SCPx and pre-nsLTP are designated by
upward, open and solid arrowheads,
respectively. Another potential cleaving site of SCPx is at position
405-406. B, immunoblotting of subcellular fractions of rat
liver was performed using antibodies to SCPx (lanes 1 and
2) and nsLTP (lanes 3-5). Lanes 1 and
3, PNS (50 µg); lanes 2 and 4,
peroxisomes (per; 10 µg); lane 5, cytosol
fraction (cyto; 50 µg). Molecular markers in kDa are on
the left. Open and solid arrowheads
indicate SCPx and nsLTP, respectively; arrow designates
46-kDa protein (mSCPx) derived from SCPx. The asterisk in
the left panel shows a nonspecific band; the dot
in lane 5 indicates pre-nsLTP.
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Morphological Analysis of nsLTP and SCPx--
Wild-type and
pex mutant CHO cells were stained using anti-nsLTP antibody.
In CHO-K1 and pex7 mutant ZPG207 defective in PTS2 import
(25), nsLTP was detected in a punctate staining pattern (Fig.
2A, a and
d), in a superimposable manner with that obtained using
anti-catalase antibody (e and h), thereby
suggesting that nsLTP was localized to peroxisomes. On the other hand,
in pex2 Z65 and pex5 ZP105 mutants defective in
PTS1 and PTS2 import (23, 24, 26, 30), nsLTP appeared to be stained
partly in a diffused pattern in the cytoplasm (Fig. 2A,
b and c), as was also the case for catalase
(f and g) (24, 26). A membrane-associated
pattern, presumably representing SCPx (see Fig.
3), is also visible by staining with
anti-nsLTP antibody in Z65 and ZP105 (Fig. 2A, b and c). These results collectively suggest that nsLTP and
SCPx cross-reacting with anti-nsLTP are imported by the PTS1, rather than PTS2, pathway. Moreover, in a recently isolated pex5
CHO cell mutant ZPG231 with a phenotype showing a defect solely in PTS2
import (31), punctate staining pattern was likewise discernible upon
cell staining using anti-nsLTP antibody, in a superimposable manner
with catalase-positive structures, hence confirming the import of nsLTP
via the PTS1 pathway (data not shown).

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Fig. 2.
Intracellular localization of nsLTP and
SCPx. A, wild-type and peroxisome biogenesis-defective
mutant CHO cells were dual-stained using antibodies to nsLTP
(a-d) and catalase (e-h). Cells were as
follows: a and e, CHO-K1; b and
f, a pex2 mutant Z65; c and
g, a pex5 mutant ZP105; d and
h, a pex7 mutant ZPG207. Magnification, ×630;
scale, 20 µm. B, CHO cells were fixed, treated
with 1% Triton X-100 (a-h) or 25 µg/ml of digitonin
(i-p), under which only plasma membranes are permeabilized,
and were double-stained with antibodies to SCPx (a-d and
i-l) and catalase (e-h and m-p).
Cells were as follows: a, e, i, and
m, CHO-K1; b, f, j, and
n, a pex2 Z65; c, g,
k, and o, a pex5 ZP105; d,
h, l, and p, a pex7 ZPG207.
Scale, 20 µm. Note that SCPx (i-l) and a
matrix enzyme catalase (m and p) were not
discernible.
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Fig. 3.
Subcellular localization of nsLTP and SCPx in
pex mutant cells. A, cytosolic
(S) and organellar (P) fractions from PNS of
wild-type and pex mutant CHO cells (8 × 105 cells each) were analyzed by SDS-PAGE and immunoblot.
Immunoblot was performed using antibodies to nsLTP (top two
panels), SCPx (middle panel), and AOx (bottom
panel). Immunoblot pattern using anti-nsLTP antibody was
essentially as in Fig. 1B (right panel); only
parts showing SCPx and nsLTP were shown as a composite.
Solid and open arrowheads indicate SCPx and
46-kDa protein (mSCPx) derived from SCPx; P and
M, a larger precursor (pre-nsLTP) and mature protein of
nsLTP, respectively. A, B, and C show
75-, 53-, and 22-kDa components of AOx; dots, nonspecific
bands (36). B, protease protection assay. PNS from
pex5 ZP105 was treated with proteinase K for 30 min on ice.
The digestion was terminated with 1 mM phenylmethylsulfonyl
fluoride. The reaction mixture was centrifuged to separate cytosolic
(S) and organellar (P) fractions, which were
analyzed by SDS-PAGE and immunoblot using antibodies to SCPx and
Pex14p. PNS fractions analyzed were from 5 × 105
cells each. PNS was mock-treated (lanes 1 and 2)
or treated with 5, 10, and 20 µg/ml of proteinase K in the absence
(lanes 3-5) or presence (lane 6) of 1% Triton
X-100. Only P fractions, each from proteinase K-treated PNS, were
loaded. In lane 6, total reaction mixture was analyzed.
C, isopycnic subcellular fractionation. PNS fractions from
CHO-K1 (upper panel) and pex2 Z65 (lower
panel) (2 × 108 cells each) were fractionated by
isopycnic ultracentrifugation on a sucrose density gradient. The
gradient was collected into 19 tubes (0.5 ml each). An equal volume (15 µl) of each fraction was analyzed by SDS-PAGE, followed by immunoblot
with antisera against SCPx, Pex14p, and Pex5p. Results are presented in
the direction of lower to higher density of sucrose, from
left to right.
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These four types of CHO cells were stained using an antibody specific
for SCPx. In CHO-K1, SCPx was detected in punctate structures that were
superimposable upon those stained for catalase, hence indicating
peroxisomal localization (Fig. 2B, a and
e). Similarly, SCPx was localized in particles in ZPG207, as
was the case for catalase, strongly suggesting that SCPx is transported
into peroxisomes (Fig. 2B, d and h).
Similar punctate structures were detected when pex5 ZPG231
was stained with anti-SCPx antibody (data not shown). In contrast, in
pex2 Z65 and pex5 ZP105 where catalase (Fig.
2B, f and g) and PTS1 proteins (data
not shown) were in the cytoplasm, SCPx was partly, if not entirely,
stained in a punctate pattern (b and c). In the
same sets of cells that were permeabilized with 25 µg/ml of
digitonin, under which plasma membranes were selectively permeabilized
and intraperoxisomal proteins were inaccessible to exogenous antibodies
(28, 32), SCPx was not detectable as a particle-associated form in any
of four types of CHO cells including wild-type, pex7,
pex2, and pex5 cells (Fig. 2B,
i-l). Catalase was not accessible in
digitonin-permeabilized CHO-K1 and pex7 ZPG207 (m
and p), whereas catalase in pex2 Z65 and
pex5 ZP105 cells was apparently visible in the cytoplasm
(n and o), slightly more vividly than the other
cell types. We interpreted these data to mean that SCPx is associated
with and possibly imported into endomembranes in pex2 and
pex5 cell mutants.
Subcellular Fractionation Analysis--
Intracellular localization
of nsLTP and SCPx was also investigated by subcellular fractionation of
CHO cells. When PNS of CHO-K1 cells was fractionated, nsLTP protein was
found in both the cytosolic fraction and organelle pellets by
immunoblotting with anti-nsLTP antibody (Fig. 3A, top
panel, lanes 1 and 2). Subcellular
localization of nsLTP and pre-nsLTP was distinct; pre-nsLTP was
exclusively in the cytosolic fraction, whereas nsLTP was in the
organelle fraction, apparently in peroxisomes (see Fig. 2). SCPx
cross-reacting with anti-nsLTP antibody was exclusively in the
organelle pellets (lane 2), presumably peroxisomes (see Fig.
2), as reported previously (2, 33). A similar distribution pattern of
pre-nsLTP, nsLTP, and SCPx was observed in pex7 ZPG207 (Fig.
3A, top panel, lanes 7 and
8). In contrast, only pre-nsLTP, but not mature nsLTP, was
present and exclusively in the cytosolic fraction from pex2
Z65 and pex5 ZP105 (top lower panel, lanes 3-6), whereas SCPx was detected by anti-nsLTP antibodies in the organelle fraction in both mutants (top upper panel,
lanes 4 and 6) as in CHO-K1 and ZPG207. Next, using
SCPx-specific antibody, SCPx and 46-kDa protein, presumably the
processed form of 59-kDa SCPx, were discernible in the organelle
fraction of both CHO-K1 and ZPG207 (Fig. 3A, middle
panel, lanes 1, 2, 7, and
8), thereby implying that SCPx is imported into peroxisomes
and partly converted to 46-kDa protein plus (presumably) nsLTP. In
contrast, there was only the 59-kDa form of SCPx detectable in membrane
pellet fractions of Z65 and ZP105 cells (middle panel,
lanes 3-6), implying that there was no processing activity
in these mutants.
AOx, a PTS1 protein, consists of 75-kDa A, 53-kDa B, and 22-kDa C
polypeptide components and exists as a hetero-oligomer comprising A2, ABC, and B2C2 (26, 34). In
normal cells, proteolytic conversion of AOx-A component to B and C
polypeptides occurs in peroxisomes (26, 35). A, B, and C components of
AOx were evident in CHO-K1 and ZPG207, indicative of normal biogenesis
of AOx (Fig. 3A, bottom panel, lanes
1, 2, 7, and 8), consistent with
our earlier observation (25). In Z65 and ZP105, AOx-A, but not the B
and C components, was detectable in cytosolic fraction at a lower level
(bottom panel, lanes 3-6), indicating failure on
the conversion of AOx, as in our earlier observation (27, 28, 36).
To determine the subcellular, as well as intraorganellar, localization
of SCPx in pex mutants such as pex5 ZP105, we
took another approach, a so-called protease protection assay. SCPx in
PNS of ZP105 cells was resistant to the treatment with exogenously added proteinase K and was sedimentable (Fig. 3B,
upper panel, lanes 1-5), whereas a peroxisomal
membrane peroxin Pex14p (37) was sensitive to the digestion
(lower panel), thereby strongly suggesting that SCPx was
inside the organelles. Treatment with Triton X-100 prior to the
protease digestion abolished the resistance, thereby confirming that
SCPx was localized in membranous vesicles. Essentially the same results
were obtained using pex2 Z65 cells (data not shown).
To confirm the findings described above with respect to the
intracellular location of SCPx, PNS from CHO-K1 and a pex2
Z65 was fractionated by isopycnic ultracentrifugation on a sucrose density gradient. From CHO-K1, both 59- and 46-kDa forms of SCPx cosedimented with Pex14p, indicating that SCPx was localized and processed in peroxisomes (Fig. 3C, upper panel).
Pex5p mostly remained nearly at the top of gradient, but a small
portion of Pex5p cosedimented with peroxisomes, indicating a largely
cytoplasmic PTS1 receptor (30, 38). In Pex2p-defective Z65 (23), 59-kDa SCPx was sedimented and distributed from the middle to the bottom of
the gradient (Fig. 3C, lower panel). Pex14p
representing peroxisomal remnants (37) sedimented to a lighter density
part of the gradient, with which a larger part of Pex5p cosedimented,
consistent with our earlier observation (30). We interpreted these
findings to mean that SCPx was localized to endomembranes, partly
associated with peroxisomal remnants, in Z65 cells. SCPx appeared to be
associated with mitochondria, as well (see Fig. 2B).
Binding to the PTS1 Receptor Pex5p--
To investigate whether
nsLTP and SCPx interact with the PTS1 receptor Pex5p, cell lysates from
CHO-K1 were incubated with GST-Pex5pL fusion protein of wild-type
Pex5pL, as well as ZP105-type mutant GST-Pex5pL-G335E severely
defective in PTS1-binding (30). Bound protein fractions were analyzed
by SDS-PAGE and immunoblot using specific antibodies. Only mature
nsLTP, not pre-nsLTP, bound to wild-type Pex5pL, but not to GST and
GST-Pex5pL-G335E, as probed with anti-nsLTP antibody, although
pre-nsLTP and nsLTP were detectable in a nearly equal amount in PNS
(Fig. 4, middle panel). This
result suggests that C-terminal AKL is recognized by Pex5p in the case of nsLTP but not of pre-nsLTP. The presequence may affect the interaction of PTS1 of pre-nsLTP with Pex5p. SCPx was likewise bound
only to normal Pex5pL (Fig. 4, top panel), where the 44-kDa processed form of SCPx lacking the C-terminal AKL was not detectable, even probed with SCPx-specific antibody (data not shown). Essentially the same results were obtained (data not shown) using GST-Pex5pS (30).
As a PTS1 control, three components of AOx hetero-oligomer (35) (see
Fig. 3A) were pulled down by normal GST-Pex5pL but barely by
GST-Pex5pL-G335E, not by GST (Fig. 4, bottom panel, lanes 3-5), consistent with our previous observation
(30).

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Fig. 4.
Binding of SCPx and nsLTP to the PTS1
receptor Pex5p. In vitro binding assay was performed by
using Pex5pL from wild-type and pex5 mutant, ZP105. GST and
fusion proteins, GST-Pex5pL and GST-Pex5pL-G335E
(GST-G335E), were incubated with cell lysates of CHO-K1
(3 × 106 cells each). After thoroughly washing,
proteins bound to glutathione-Sepharose were analyzed by SDS-PAGE. SCPx
and nsLTP were detected by immunoblot using anti-nsLTP antibody; AOx
was assessed as a control PTS1 protein. Arrowhead indicates
SCPx; P and M show pre-nsLTP and mature protein
of nsLTP, respectively. A, B, and C
designate polypeptide components of AOx. Lanes 1 and
2, cytosolic (S) and organelle (P)
fractions from PNS of CHO-K1 (one-tenth aliquot each used for the
pull-down assay); lanes 3-5, bound proteins to GST and
GST-Pex5p fusion proteins as indicated. Note that SCPx and nsLTP, but
not pre-nsLTP, specifically bound to Pex5pL.
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Intracellular Localization of GFP Fusion Protein--
To elucidate
whether the presequence of pre-nsLTP and/or C-terminal AKL function as
a sufficient PTS, we constructed two types of GFP-fusion proteins, one
fused N-terminally to the 25-amino acid residues including the 20-amino
acid presequence plus 5 downstream-residues of pre-nsLTP and
C-terminally linked to AKL (termed pre-GFP-AKL), and the other fused
only to the presequence at the N terminus (pre-GFP) (Fig.
5A). These chimeras were
transiently expressed in CHO-K1, pex2 Z65, pex5
ZP105, and pex7 ZPG207 cells. In CHO-K1, pre-GFP-AKL was
detected in particulates and colocalized with catalase, hence
indicating that pre-GFP-AKL was targeted to peroxisomes (Fig.
5B, a and e). pre-GFP-AKL was likewise
in particles, in catalase-positive but PTS2-negative "peroxisomes"
in ZPG207 (25) (Fig. 5B, d and h). In
contrast, pre-GFP-AKL appeared to remain partly in the cytoplasm, like
catalase, in pex2 and pex5 mutants (Fig.
5B, b, c, f, and
g). In these mutants, pre-GFP-AKL was also discernible in a
membrane-associated form as seen for nsLTP and SCPx (see Fig. 2,
A and B), which was superimposable on that
assessed by cell staining using antibody to malate dehydrogenase, a
mitochondrial marker enzyme (Fig. 5C). Accordingly, the data
strongly suggested that pre-GFP-AKL was mostly localized to
mitochondria in pex2 and pex5 mutants. Taken
together, pre-GFP-AKL was transported to peroxisomes in a
PTS1-dependent and Pex5p-mediated pathway in normal and
PTS2-import-defective pex7 cells. Moreover, we likewise investigated whether the presequence of pre-nsLTP functions as a
topogenic signal. pre-GFP was detected in a superimposable manner with
malate dehydrogenase-positive particles, presumably mitochondria, in
both wild-type and pex2 CHO cells (Fig. 5D),
suggesting that the 20-amino acid presequence functions per
se as a mitochondrial targeting sequence.

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Fig. 5.
Expression of GFP tagged with both N-terminal
presequence and C-terminal tripeptide AKL of pre-nsLTP or only with
N-terminal presequence. A, schematic representation of
GFP fusion constructs. pre-GFP-AKL, C-terminally tripeptide AKL
(PTS1)-tagged GFP was N-terminally fused at DNA level with the
N-terminal sequence of pre-nsLTP (residues 1-25, taking initiator Met
as I) including the entire presequence 1-20; pre-GFP, AKL
was deleted from pre-GFP-AKL. B, CHO cells were transfected
with cDNA encoding pre-GFP-AKL. Cells were as follows: a
and e, CHO-K1; b and f, a
pex2 Z65; c and g, a pex5
ZP105; d and h, a pex7 ZPG207.
Expressed GFP fusion protein was monitored by fluorescence
(a-d). Peroxisomes were assessed by immunostaining of
catalase (e-h). Scale, 20 µm. C,
pex2 Z65 and pex5 ZP105 cells were transfected
with cDNA for pre-GFP-AKL. GFP fusion protein was assessed by
fluorescence (a and b). Mitochondria were
detected by staining with anti-malate dehydrogenase
( -MDH) antibody (c and
d). Scale, 20 µm. D, CHO-K1 and a
pex2 Z65 were transfected with cDNA for pre-GFP
cDNA. Detection of GFP and mitochondria was done as in
C. Scale, 20 µm.
|
|
Interaction of pre-GFP-AKL with Pex5p--
The expression level of
pre-GFP-AKL and pre-GFP was also verified by immunoblot of the cell
lysates. Both proteins were detected with an expected size and at a
similar level in wild-type CHO-K1 and a pex2 Z65 (Fig.
6A, lanes 2,
3, 5, and 6). As a control GFP-AKL (28) was also expressed, which was detectable in both types of cells
(lanes 1 and 4), and was targeted to peroxisomes
in CHO-K1 but remained in the cytoplasm in Z65 (data not shown), in
good agreement with our earlier observation (28). The distinct mobility in SDS-PAGE of these three GFP fusion proteins suggested that the
presequence of pre-GFP-AKL and pre-GFP was not cleaved off, even after
imported to peroxisomes and mitochondria, respectively. The processing
site of these chimera may not be recognized by a potential processing
protease. Next, we investigated whether pre-GFP-AKL interacts with
Pex5p. Control GFP-AKL bound to GST-Pex5pL, but not to GST (Fig.
6B, lanes 1-3), consistent with peroxisomal localization (data not shown) (28). pre-GFP-AKL, but not pre-GFP, from
CHO-K1 cell-lysates was likewise pulled down by GST-Pex5pL, not by GST
(Fig. 6B, lanes 4-9), thereby indicating that,
in contrast to pre-nsLTP, the PTS1 of pre-GFP-AKL was readily
recognized by Pex5p, in agreement with morphological findings (see Fig.
5B).

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|
Fig. 6.
Binding of pre-GFP-AKL to Pex5p.
A, expression level of GFP-AKL (lanes 1 and
4), pre-GFP (lanes 2 and 5), and
pre-GFP-AKL (lanes 3 and 6). CHO-K1 (lanes
1-3) and Z65 (lanes 4-6) cells (3 × 104) were transfected with each cDNA construct
indicated at the top. Cell lysates were analyzed by SDS-PAGE
and Western blot using anti-GFP antibody. B, binding assay
of GFP-AKL, pre-GFP, and pre-GFP-AKL to Pex5p was done using
cell-lysates of GFP-AKL-, pre-nsLTP-, and pre-GFP-AKL-expressing CHO-K1
(1 × 106 cells each), as in Fig. 4. Components added
in the GST pull-down assay were indicated at the top. GFP
fusion protein was detected by Western blot using anti-GFP antibody.
One-tenth aliquot of each input was loaded in lanes 1,
4, and 5.
|
|
 |
DISCUSSION |
pre-nsLTP can be classified as a unique protein among a number of
peroxisomal proteins with respect to its topogenic sequence. The
cleavable presequence locates at the N terminus, similar to PTS2 of
several peroxisomal enzymes such as 3-ketoacyl-CoA thiolase (20, 21),
and the C terminus carries a typical PTS1 tripeptide motif, AKL.
Moreover, the amino acid sequence, -AAPT
SS- (-, continuing to
adjacent residues), near the processing site of pre-nsLTP to form mature nsLTP resembles the sequence of PTS2-thiolase, -AAPC
SS- (-, continuing to adjacent residues), implying that pre-nsLTP and PTS2 proteins may share a potential, yet unidentified processing protease present in peroxisomes. The nsLTP presequence is relatively positive-charged like PTS2, but it does not contain the consensus PTS2
sequence -(R/K)(L/V/I)X5(H/Q)(L/A)-. This
implies that the presequence may be a novel-type PTS. We investigated
in the present work which of two potential PTS sequences destines
pre-nsLTP to its final intracellular location in vivo. With
the use of several CHO pex mutants, import of pre-nsLTP was
verified. pre-nsLTP was affected in import in pex2 and
pex5 mutants with a phenotype showing import defect of both
PTS1 and PTS2 proteins, where pre-nsLTP remained in the cytosol. Other
groups of investigators reported that nsLTP is likely to be degraded or
barely detectable in ZR82, a pex2 CHO mutant (33), as well
as in fibroblasts from a patient with Zellweger syndrome (12), although
pre-nsLTP was not fully described. In pex7 cell mutant
impaired in PTS2 import, pre-nsLTP was translocated as efficiently as
in the wild-type CHO-K1. Transport of pre-GFP-AKL was likewise
re-established in wild-type and pex7 mutant CHO cells,
consistent with the morphological phenotype as assessed for pre-nsLTP.
Taken together, we conclude that pre-nsLTP is imported by the
Pex5p-mediated PTS1 pathway.
To our surprise, pre-GFP-AKL was targeted to mitochondria when
expressed in pex2 and pex5 mutants. These results
imply that pre-GFP-AKL behaves differently from pre-nsLTP; PTS1 of
pre-nsLTP could not be recognized by Pex5p in vitro, whereas
AKL of pre-GFP-AKL readily interacted with Pex5p. Despite such
biochemical properties, pre-nsLTP is transported to peroxisomes by a
Pex5p-dependent pathway in vivo. Moreover, in
mutant cells absent from Pex5p, pre-nsLTP remains in the cytoplasm,
whereas pre-GFP-AKL is readily transported to mitochondria at least in
an over-expression system. The mitochondrial topogenic activity appears
to be interfered with in the presequence of pre-nsLTP. Accordingly, the
PTS1 and presequence of pre-nsLTP are most likely to be mutually
regulated by unknown mechanisms. One possibility includes modulation of
PTS1 of pre-nsLTP by direct steric effect of the presequence or that
mediated by a cytosolic factor. Alternatively, PTS1 of pre-nsLTP may be
partly modified, whereby a portion of total pre-nsLTP no longer
interacts with Pex5p and remains in the cytoplasm. In the case of
pre-GFP-AKL, the tripeptide AKL is more likely to be exposed to the
surface of the molecule, thereby being readily recognized by Pex5p. The presequence may be unable to modulate the PTS1, possibly because of the
configuration of this GFP chimera. Furthermore, we identified the
presequence as a mitochondrial targeting signal, based on the
observation that pre-GFP was targeted to mitochondria in normal and
mutant CHO cells.
It is noteworthy that the N-terminal sequence encompassing the 20-amino
acid presequence plus Ser at position 21 was predicted as a
mitochondrial targeting sequence (44% probability) using a recognition
program, PSORT II, minimally requiring 21 amino acid residues to be
analyzed, where the prediction also included cytoplasmic (26%) and
peroxisomal (4%) localization. Secondary structure analysis of the
20-amino acid presequence using a Chou-Fasman program predicted an
-helix between residues at 3-10 and
-sheet structure between the
residues 10-17. These characteristics may contribute as a
mitochondrial import signal. Therefore, it is tempting to infer that
the presequence may interact with the C-terminal PTS1 and regulate
peroxisomal transport of pre-nsLTP, possibly in a concerted manner with
other cytosolic factors such as chaperones. How the PTS1 and
mitochondrial targeting signal-like presequence are regulated in
biogenesis of nsLTP in vivo remains to be defined.
pre-nsLTP is rather stably present in the cytosol as assessed by
subcellular fractionation study and morphological analysis. The
modulated presequence may function in vivo as a signal for pre-nsLTP to be a cytosolic protein. If synthesized pre-nsLTP remains
in the cytoplasm, nsLTP in peroxisomes may be all derived from SCPx
instead of pre-nsLTP by proteolytic conversion. Although we can not
exclude this possibility, this may be less likely based on the findings
in pulse-chase experiments, where processing of pre-nsLTP to nsLTP was
noted (2, 12). The physiological consequences of pre-nsLTP in the
cytosol, nearly at an equal level as nsLTP in peroxisomes in normal CHO
cells, also remain to be investigated. The level of cytosolic pre-nsLTP
may vary, depending on the cell types from various tissues and organs
that are involved in lipid metabolism and steroid biosynthesis (1, 14).
It is also notable that a role of nsLTP and SCPx in peroxisomal
-oxidation of phytanic acid was implicated from the study using
nsLTP/SCPx gene null mice (39).
SCPx is exclusively localized in peroxisomes where it is converted to
the 46-kDa fragment and nsLTP. SCPx is also transported with use of
C-terminal AKL via a Pex5p-mediated PTS1 pathway. In
PEX5-deficient mutant cells, SCPx is associated with
endomembranes such as peroxisomal remnants and mitochondria. It is
noteworthy that not only the 59-kDa form but also the 46-kDa fragment
are enzymatically active as a branched-chain fatty acid thiolase (15, 17, 40), as revealed using specimens from patients with
peroxisome-defective disorders such as Zellweger syndrome. Localization
of SCPx on endomembranes may be required for such enzymatic activity of
the unprocessed form. However, its underlying mechanisms are presently unclear. Exact cleavage sites initially at position 404-405 or 424-425 or both (see Fig. 1A), as well as physiological
significance of this intraperoxisomal processing of SCPx, are not well
understood at present. Interestingly, P-44 with thiolase activity and
ZK892.2 apparently corresponding to the 46-kDa form of SCPx and nsLTP in mammals, respectively, have both been identified in
Caenorhabditis elegans (41), whereas an orthologue of 59-kDa
SCPx has not been detected. Whether accumulation of SCPx in
protease-resistant form in the membrane fraction in pex5 and
pex2 mutants, as noted in this work, is physiologically
relevant or related to clinical phenotypes of peroxisome biogenesis
disorders also remains to be defined.
 |
ACKNOWLEDGEMENTS |
We thank S. Tamura for advice, M. Honsho
and N. Thomas for comments, and the other members of our laboratory
for discussion.
 |
FOOTNOTES |
*
This work was supported in part by a CREST grant (to Y. F.)
from the Japan Science and Technology Corporation and Grants-in-Aid for
Scientific Research 08557011, 09044094, 12308033, 12557017, and
12206069 (to Y. F.) from The Ministry of Education, Science, Sports,
and Culture.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biology,
Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.:
81-92-642- 2635; Fax: 81-92-642-4214; E-mail:
yfujiscb@mbox.nc.kyushu-u.ac.jp.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007730200
 |
ABBREVIATIONS |
The abbreviations used are:
nsLTP, nonspecific
lipid transfer protein;
pre-nsLTP, larger precursor of nsLTP;
AOx, acyl-CoA oxidase;
CHO, Chinese hamster ovary;
GFP, green fluorescent
protein;
PAGE, polyacrylamide gel electrophoresis;
PNS, post-nuclear supernatant fraction;
PTS(s), peroxisome targeting signal(s);
SCPx, sterol carrier protein x;
GST, glutathione
S-transferase.
 |
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