From the Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581 and Core Research for Evolution Science Technology, Japan Science and Technology Corporation, Tokyo 107-0013, Japan
Received for publication, April 18, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Human 34-kDa peroxisomal membrane protein
(PMP34) consisting of 307 amino acids was previously identified as an
ortholog of, or a similar protein (with 27% identity) to the,
423-amino acid-long PMP47 of the yeast Candida boidinii. We
investigated membrane topogenesis of PMP34 with six putative
transmembrane segments, as a model peroxisomal membrane protein. PMP34
was characterized as an integral membrane protein of peroxisomes.
Transmembrane topology of PMP34 was determined by differential
permeabilization and immunofluorescent staining of HeLa cells
ectopically expressing PMP34 as well as of Chinese hamster ovary-K1
expressing epitope-tagged PMP34. As opposed to PMP47, PMP34 was found
to expose its N- and C-terminal parts to the cytosol. Various deletion
variants of PMP34 and their fusion proteins with green fluorescent
protein were expressed in Chinese hamster ovary-K1 and were verified
with respect to intracellular localization. The loop region between transmembrane segments 4 and 5 was required for the
peroxisome-targeting activity, in which Ala substitution for basic
residues abrogated the activity. Three hydrophobic transmembrane
segments linked in a flanking region of the basic loop were essential
for integration of PMP34 to peroxisome membranes. Therefore, it is
evident that the intervening basic loop plus three transmembrane
segments of PMP34 function as a peroxisomal targeting and
topogenic signal.
The peroxisome is a model system for addressing protein traffic
and membrane biogenesis, especially where it is linked to human
neurological and metabolic diseases, called peroxisome biogenesis disorders. Import of matrix proteins into peroxisomes has been investigated to a greater extent at the molecular and cellular levels
(1, 2). Peroxisomal matrix proteins are synthesized on free polysomes,
and most contain peroxisome targeting signals type 1 or 2, called
PTS11 or PTS2. Cytosolic
receptors for PTS1 and PTS2, Pex5p and Pex7p, respectively, function in
the transport of cargo proteins to peroxisomes, by docking with the
convergent membrane peroxin, Pex14p (3-5). In contrast, molecular
mechanisms involved in membrane protein transport and membrane assembly
of peroxisomes are not well understood. Peroxisomal integral membrane
proteins (PMPs), such as 22-kDa PMP (PMP22) (6), PMP70 (7), and Pex2p
(8) of rat liver are also synthesized on free polysomes.
Post-translational import of PMP22 and PMP70 were shown in
vitro (9, 10). It is noteworthy that Pex16p and Pex2p are
N-glycosylated in the yeast Yarrowia lipolytica
(11). PTS of membrane protein, termed mPTS, was previously suggested
for PMP47 with six putative transmembrane segments of the yeast
Candida boidinii (12). The loop region between the fourth
transmembrane segment (TM4) and TM5, which was enriched in positively
charged amino acids was functional as a PTS when fused to cytosolic
proteins (12). It is also noteworthy that an internal region, including
the predicted TM3, of rat PMP70, a six-TM protein, is essential for the
peroxisomal localization (10). The N-terminal amino acid residues at
positions 1-40, which are relatively enriched in positive charged
residues, were recently shown to be required for translocation of
Pex3p, an integral membrane peroxin, to peroxisomes in mammals (13-15)
and yeast (16, 17). Accordingly, to delineate mPTS and to establish a
general paradigm for topogenesis of PMPs, more information is required.
In the present study, we have chosen PMP34 as a model protein, to
address these issues. PMP34 was recently cloned by expressed sequence
tag data base search using C. boidinii PMP47 and was shown
to be localized to peroxisomes (18). We found that PMP34 is an integral
membrane protein of peroxisomes. In contrast to PMP47, PMP34 exposes
both N- and C-terminal parts to the cytosol. We also identified the
loop region between TM4 and TM5 as a potential mPTS. This loop plus
three TMs were essential for targeting and integration of PMP34. These
results provide the first evidence for a functional mPTS for the
topogenesis of a membrane protein that spans membranes multiple times
in mammalian peroxisomes.
Reagents and Biochemicals--
Restriction enzymes and DNA
modifying enzymes were purchased from Nippon Gene (Tokyo, Japan),
Toyobo (Tokyo, Japan), and Takara (Tokyo, Japan). Fetal calf serum and
Ham's F-12 were from Life Technologies, Inc. Anti-PMP34 antibody was
raised in rabbits by immunizing with synthetic peptide comprising the
C-terminal, 19-amino acid sequence of human PMP34, supplemented with
cysteine at the N terminus that had been linked to keyhole limpet
hemocyanin (19). Rabbit antibody against influenza virus hemagglutinin
(HA) was likewise raised using synthetic peptide,
CYPYDVPDYASLRS-NH2. Guinea pig antibody to human catalase
(Sigma) was raised by conventional subcutaneous injection. We also used
rabbit antibodies to PTS1 peptide (20), acyl-CoA oxidase (AOx) (21),
and C-terminal 19-amino acid residues of rat Pex14p (4). Rabbit
antibody to green fluorescent protein (GFP)
(CLONTECH) and mouse monoclonal antibodies to flag
(M2) and HA (12CA5) (Sigma) were purchased.
Cell Culture--
CHO-K1 and HeLa cells were cultured in Ham's
F-12 medium and RPMI 1640, respectively, both supplemented with 10%
fetal calf serum, under 5% CO2, 95% air (21).
Isolation and Epitope Tagging of HsPMP34--
Human PMP34
cDNA (HsPMP34) was cloned by PCR-based technique on the
human liver cDNA library in pCMVSPORT (Life Technologies, Inc.)
(22), using a set of primer, HsPMP34F and HsPMP34R (Table I). Six
independent cDNA clones were sequenced, all showing that nucleotide
residue at position 315 in a codon (CTG) for
Leu105 was G instead of CTC reported by Wylin
et al. (18). HsPMP34 cDNA was cloned into the
NotI-SalI site in pUcD2HygSR Construction of PMP34 Variants--
N-terminal truncation
mutants of PMP34 were generated by PCR, using as forward primers,
Alanine substitution in the fourth loop domain between TM4 and TM5 of
PMP34 was done by PCR using as a template
pGEM/T-easy·204Ter and a forward primer HsPMP34F and a
reverse primer 6Ar, 4Ar, 3Ar, or Ar. Construction 2A was done by PCR
using as a template pGEM/T-easy·6A204Ter and a forward
primer HsPMP34F and a reverse primer 2Ar. GFP tagging was performed as
described above.
Expression of PMP34 and Its Derivatives in HeLa and CHO-K1
Cells--
DNA transfection was done using LipofectAMINE (Life
Technologies, Inc.), as described (23). The cells were cultured for 36 h after transfecting the plasmid into the cells. Cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100, and peroxisomes were visualized by indirect immunofluorescence light microscopy, as described (26). Antigen-antibody complexes were
detected under a Carl Zeiss Axioskop FL microscope, using fluorescein
isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham
Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit
immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat
antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG
(Leinco Technologies). GFP was directly observed by fluorescent
microscopy with the use of the same filter for FITC after fixation
(27). Flag-PMP34-HA was detected using rabbit anti-HA antibody and
mouse anti-flag antibody, in cells that had been fixed as above and
then permeabilized with either 25 µg/ml digitonin or 1% Triton X-100
(23, 28).
HeLa cells were transfected with pUcD2HygSR Protease Sensitivity Assay--
HsPMP34-transfected
CHO-K1 cells (1 × 107) were homogenized in 0.5 ml of
a homogenizing buffer: 0.25 M sucrose, 25 mM
ammonium carbonate, pH 7.4, 20 µg/ml each of leupeptin and antipain,
and 500 units/ml aprotinin, by 10 strokes of an Elvehjem-Potter
homogenizer. A postnuclear supernatant (PNS) fraction was prepared by
centrifugation of homogenates at 750 × g for 5 min.
The PNS from 1 × 106 cells was treated with 2 µg of
Staphylococcus aureus V8 protease (Roche Molecular
Biochemicals) at 25 °C for 1.5 h in 0.5 ml of the homogenizing
buffer, in the absence or presence of 1% Triton X-100. The reaction
was terminated by precipitation using trichloroacetic acid, and whole
proteins were then analyzed by SDS-polyacrylamide gel electrophoresis
(PAGE) and immunoblot.
Other Methods--
Liver peroxisomes were isolated from a normal
rat, as described (29). Western blot analysis was done using primary
antibodies and a second antibody, donkey anti-rabbit IgG antibody
conjugated to horseradish peroxidase (Amersham Pharmacia Biotech).
Antigen-antibody complex was visualized with ECL Western blotting
detection reagent (Amersham Pharmacia Biotech).
Characterization of HsPMP34 Protein--
The intracellular
localization of PMP34 was determined by ectopic expression of
epitope-tagged HsPMP34 and immunofluorescence microscopy. In
wild-type CHO-K1 cells expressing PMP34 tagged with HA at its C
terminus, PMP34 was detected as a punctate staining pattern using
anti-HA antibody. The staining pattern was superimposable with that
obtained using anti-Pex14p antibody, thereby suggesting that PMP34-HA
was targeted to peroxisomes (Fig.
1A). Similar results were
obtained when HsPMP34 was expressed in HeLa cells and
stained with anti-PMP34 peptide antibody (see Fig. 1C). The
rabbit antibody raised to the C-terminal peptide of human PMP34
specifically reacted with an endogenous, single protein with an
apparent molecular mass of ~34 kDa in immunoblot of rat liver
peroxisomes (Fig. 1B, left), confirming that
PMP34 is a bona fide protein of peroxisomes. By sequence
similarity to C. boidinii PMP47 (12, 18), HsPMP34 is likely to be an integral membrane protein with six putative transmembrane segments. The integrity of PMP34 in peroxisomal membranes
was verified by extraction with 0.1 M sodium carbonate, pH
11.3 (30). PMP34-HA expressed in CHO-K1 cells was not extracted with
sodium carbonate (Fig. 1B, right) and was
recovered in membrane fraction at a similar level as in postnuclear
supernatant fraction (data not shown). Pex14p, a peroxisomal membrane
peroxin, was likewise in membrane pellet, whereas a matrix enzyme AOx
was in soluble fraction. These data thereby indicated that PMP34 is
integrated into membranes.
Next, we determined transmembrane topology of PMP34 by the differential
permeabilization/immunofluorescence microscopy method (28, 31). HeLa
cells were transfected with HsPMP34. Upon treatment with 1%
Triton X-100, which solubilizes all cellular membranes, a punctate
staining pattern was observed using anti-PMP34 C-terminal peptide
antibody, in a superimposable manner with catalase presumably representing peroxisomes (Fig. 1C, a and
c). Similar morphological pattern, PMP34-staining, was
observed after permeabilization with 25 µg/ml digitonin, which
selectively permeabilized plasma membranes (28, 31) (Fig.
1C, b), whereas none of the cells were stained with anti-catalase antibody under the same condition (Fig.
1C, d), indicating that peroxisomal matrix
proteins are not accessible to antibody. Taken together, C-terminal
part of PMP34 is most likely to be exposed to the cytosol. Moreover,
the C-terminal part of PMP34 in HsPMP34-overexpressing
CHO-K1 was likewise detected by the digitonin treatment, although
requiring much higher concentration of digitonin under which conditions
matrix proteins still remained inaccessible to antibodies (data not
shown). We do not know the reason why PMP34 was visible only at higher
concentration of digitonin in this particular case, as compared with
that normally permeabilizing plasma membranes (4, 23). Membrane
topology of PMP34 was also determined using N-terminally flag- and
C-terminally HA-tagged PMP34. When flag-PMP34-HA-transfected
CHO-K1 cells were permeabilized with 25 µg/ml digitonin, a punctate
staining pattern was observed using both anti-flag and anti-HA
antibodies (Fig. 1D, b and f). In
contrast, PTS1 proteins were not detectable under the same condition,
indicating that lumenal proteins are not accessible to the antibody
(Fig. 1D, d). Upon treatment with Triton X-100, PTS1 proteins as well as PMP34 were discernible in numerous particles, peroxisomes (Fig. 1D, a, c, and
e). Therefore, it is apparent that N- and C-terminal
portions are exposed to the cytosol. Of note, the topology of PMP34
contrasts with that of PMP47, which exposes both terminal parts to
matrix of peroxisomes (12).
Furthermore, PMP34 expressed in CHO-K1 cells was resistant to the
treatment with exogenously added S. aureus protease V8, as
verified using PNS fraction, under which condition Pex14p was digested
(Fig. 1E, left panel). Matrix enzyme AOx was also
fully protected from the digestion. Both PMP34 and AOx were no longer discernible after protease treatment in the presence of Triton X-100
(Fig. 1E, lane 3). These results were interpreted
to mean that multiple sites, C-terminal side of Glu, for cleavage by V8 protease locate inside of peroxisomes (Fig. 1E, right
panel). It is conceivable that three potential cleavable Glu sites
may not be readily attacked by the protease, probably owing to their location close to the transmembrane-spanning segments. The data together support the notion described above that the N- and C-terminal parts of PMP34 are exposed to the cytosol.
The Fourth Loop Domain of PMP34 Contains Peroxisome Membrane
Targeting Signal--
Although transmembrane topology of PMP34 is
opposite to that of CbPMP47, amino acid sequence of the
fourth intervening-loop region of PMP34 is similar to the one
containing mPTS of PMP47 (12) (see Fig. 3A). To search for
peroxisome targeting signal of PMP34, various mutants with deletion
either from N or C terminus were constructed (Fig.
2A). Mutants
Next, we constructed two other types of PMP34 mutants truncated in the
C-terminal region and analyzed their intracellular localization. A
deletion mutant, 204HA, truncated from the C terminus to the fifth
transmembrane segment was localized to peroxisomes in CHO-K1, as
assessed by colocalization with Pex14p (Fig. 2B, m and n). Moreover, the expressed 204HA protein
showed the membrane topology exposing the HA-tagged C terminus to the
cytosol and was resistant to the sodium carbonate treatment (data not
shown), hence indicating that 204HA was properly targeted and
integrated to peroxisome membranes. In contrast, cytoplasmically
diffused staining was observed in the cells expressing 186HA truncated from C terminus to fourth loop segment (Fig. 2B,
l). Essentially the same results were obtained using GFP as
a reporter fusion protein; a fusion protein, 204GFP, was found in
peroxisomes (Fig. 2B, o and p), while
186GFP was not localized to peroxisomes (data not shown).
We further investigated transmembrane topology of C-terminal portion of
Positively Charged Region of the Fourth Loop Functions as a
Peroxisome Targeting Signal--
The fourth loop domain of PMP34 has
positively charged amino acids at positions 190, 191, 195-197, and 199 (Fig. 3A). The positively
charged amino acid cluster in the fourth loop of CbPMP47 was
shown to be sufficient for localizing soluble reporter proteins to
peroxisomes in yeast (12). To investigate whether the sequence enriched
in basic amino acids in the fourth loop of PMP34 functions as an mPTS,
we first replaced all basic amino acids to Ala of this region in
204GFP, termed 6A204GFP (Fig. 3B). In the cells expressing
6A204GFP, GFP fluorescence was diffused to the cytosol, suggesting that
positively charged amino acids are required for peroxisome targeting
(Fig. 3C, a). To determine which amino acid is
important for the peroxisome-targeting function, we subdivided the
fourth loop domain into two parts and changed basic amino acids in
respective parts to Ala. Mutants 4A204GFP and 3A204GFP were partially
localized to peroxisomes as well as in the cytoplasm (Fig.
3C, b and c). In contrast, 2A204GFP
and A204GFP was localized to peroxisomes (Fig. 3C,
d and e), as efficiently as 204GFP. Four chimera
proteins, 4A204GFP as well as 3A204GFP localized to peroxisomes, 2A204GFP, and A204GFP, showed the same membrane topology, exposing C-terminal GFP to the cytoplasm (data not shown). Therefore, it is more
likely that KR in the first portion and KKRMK in the second part
function as mPTS and that the latter half of the loop is the most
important.
The Fourth Loop Is Not Sufficient for Peroxisomal
Localization--
Dyer et al. (12) reported that the last
12 amino acids of the fourth loop domain of CbPMP47 was
sufficient for targeting to peroxisomes. So we fused the fourth loop
domain of PMP34 to N- or C terminus of GFP and analyzed its sufficiency
for peroxisome localization. To determine whether the fourth loop
functions as a sufficient information for peroxisomal targeting, a
fusion protein 187/204GFP, the loop domain fused to the N terminus of
GFP was expressed in CHO-K1, and found not to be localized to
peroxisomes (Figs. 4A and
4B). Contrary to this, another fusion protein, GFP187/204HA, was targeted to mitochondria (Fig. 4B), although the
positively charged loop in the fusion construct was located at position
distinct from a general, N-terminal mitochondrial targeting sequence.
These results demonstrated that the fourth loop domain of PMP34 is
necessary for transport of PMP34 to peroxisomes, but not sufficient for integration into peroxisomal membranes.
Three Transmembrane Segments of PMP34 Are Required for
Integration--
204GFP and
To ask if three TMs of PMP34 are also required in the N-terminal
portion of the fourth loop, we expressed 30/204GFP carrying three TMs,
TM2-TM4, and analyzed its intracellular localization. The 30/204GFP
was detected mostly in particles (Fig. 5B, c). In the GFP-positive cells, punctate structures were only partly
superimposable with those stained using Pex14p antibody (Fig.
5B, c and d). These results suggest
that the presence of simply three TMs upstream of the loop is not
sufficient for localization, rather implying that TMs from the first to
the third or the fourth to the sixth are required for integration to
peroxisomal membranes. To confirm this results, a fusion protein
86/273GFP containing the third to fifth TMs of PMP34 was expressed in
CHO-K1. Localization of 86/273GFP was not in peroxisomes (Fig.
5B, f). A chimera 86/230GFP, where GFP was fused
to immediately downstream of TM5, showed very similar GFP fluorescence
pattern as 86/273GFP (data not shown). Taken together, it is most
likely that the fourth loop and three TMs, comprising either TM1-TM3
or TM4-TM6, coordinately function as peroxisomal membrane-targeting
and integration information.
To elucidate the molecular mechanisms involved in topogenesis of
PMPs, we selected PMP34 as a model PMP in the present study. We first
determined the transmembrane topology of PMP34 by expressing the
full-length human PMP34 in HeLa cells as well as epitope-tagged PMP34
in CHO-K1 cells. From several lines of morphological assessment using a
combination of ectopic expression and the differential permeabilization
method, we concluded that PMP34 exposes both N- and C-terminal portions
to the cytoplasm. The orientation of PMP34 contradicts that of PMP47, a
potential yeast ortholog of PMP34, of which both terminal regions
reside in the matrix side of peroxisomes (12). One possible
interpretation of these observations would be that PMP34 is not an
ortholog of CbPMP47, at least with respect to the
transmembrane topology; another one is that it is a homolog but the
membrane topology is distinct. Otherwise, PMP34 is closely related to
PMP47 but a distinct protein. Physiological function of PMP34 has not
been elucidated, while PMP47 was recently suggested to be a transporter
of ATP required in activating middle-chain fatty acids with CoA (32).
We noted that PMP34 contains in the first loop region a highly
conserved sequence,
PX(D/E)XX(K/R)(X20-30)(D/E)G(X4)X(K/R)G, found in the extramembrane loop of mitochondrial carrier proteins (33,
34). PMP34 may also be a transporter like others, such as a
Ca2+-dependent peroxisomal transporter exposing
both terminal regions to the cytoplasm (35).
We demonstrated that the hydrophilic loop between TM4 and TM5 of PMP34,
similar to that of PMP47, is required for transport to peroxisomes but
is not sufficient for integration. Instead, this basic loop plus at
least three TMs, such as TM1-TM3, are likely to be responsible for the
proper localization of PMP34. In the assay system we used, it was very
difficult to discriminate between the activities of targeting and
integration. We concluded, however, that the TM4-TM5 loop domain
functions as mPTS, based on the following observations. 1) A PMP34
mutant 186HA, including TMs 1-4 but devoid of the fourth loop domain,
was not transported to peroxisomes; 2) a mutant PMP34-GFP fusion,
6AGFP, in which all of the six basic amino acids were substituted by
Ala, was completely mislocalized to the cytosol; 3) the fourth loop
domain faces to the cytosol; but it is unlikely that this loop can
translocate through the membrane into the matrix side and finally
re-translocate back to the cytoplasmic face. Together, these findings
strongly suggest that the fourth loop domain functions as an mPTS.
The fourth loop region of PMP47 is enriched in positively charged amino
acids and functions as a mPTS (12). The region responsible for
peroxisomal targeting of other membrane proteins, including Pex3p and
PMP70, has been searched for. N-terminal region, residues at positions
1-40, of the membrane peroxin Pex3p was shown to be necessary and
sufficient for peroxisomal targeting in mammals (13-15) and yeast,
Pichia pastoris (16) and Hansenula polymorpha (17). The highly conserved residues at 9-15, LKRHKKK, of human and
H. polymorpha Pex3p was recently suggested as an mPTS (14, 17). This sequence is very similar to that of the basic loop in
CbPMP47 (12), thereby suggesting that the mechanisms of
targeting Pex3p and PMP47 may resemble. Imanaka et al. (10)
demonstrated that the N-terminal sequence encompassing one third of the
full-length rat PMP70 was necessary for in vitro targeting
and integration. In this regard, Sackesteder et al. (36)
very recently showed the N-terminal residues at 1-124 of human PMP70
are targeted to peroxisomes in vivo. It is also noteworthy
that positively charged amino acid residues are noted immediately
downstream of the TM1 (residues at 28-42) as well as upstream of the
TM3 (residues at 117-124).
Between PMP34, PMP47, PMP70, and Pex3p, the cluster enriched in
relatively positively charged amino acids which located flanking region
of hydrophobic segment is a common feature. However, no conserved amino
acid sequence is observed. In our Ala-scanning analysis of the basic
loop of PMP34, the peroxisomal targeting activity decreased as the
number of mutated residues in the basic amino acids increased. A mutant
PMP34-GFP fusion, 4AGFP, in which four of the six basic amino acids
were substituted by Ala, was partially localized to peroxisomes.
Another mutant, 204Rev-GFP, where five amino acid residues at 195-199
were reversed, was localized only to peroxisomes (data not shown).
Thus, the fourth basic loop domain is most likely to function as mPTS,
despite there being apparently no need to specify the positively
charged amino acids. Whether such basic amino acids are prerequisite
for targeting of other PMPs remains to be determined. Similarly, a
conformational requirement is postulated for the mPTS of PMP47 (12). In
contrast to PMP47, the fourth loop region of PMP34 was predicted to
form an We conclude from the following observations that three TMs 1-3 or 4-6
are essential for integration of PMP34 to peroxisomes. 1) PMP34 mutants
with only two TMs were not localized to peroxisomes; 2) fusion
proteins, 204GFP and
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Synthetic oligonucleotide primers used
(23). Tagging of
epitopes, flag, and tandem HA (HA-HA) to the N and C terminus,
respectively, of human PMP34 was conducted as follows. The full length
of HsPMP34 was amplified using a pair of primers, HsPMP34F
and PMP34RNhe. The PCR product was digested with NotI and
NheI, then ligated into the NotI-NheI
sites, upstream of a double-HA tag sequence, of
pUcD2HygSR
·PEX16-HA (24).
NcoI-SalI fragment of PMP34-HA was
replaced into the NcoI-SalI sites of pBluescript
SK(
)flag-PEX13 (25), to construct
flag-PMP34-HA. The NotI-SalI fragment
of pBluescript SK(
)flag-PMP34-HA was subcloned into
pUcD2HygSR
, termed pUcD2HygSR
·flag-HsPMP34-HA. All
plasmid constructs were assessed by sequence analysis.
N30F,
N125F,
N186F, or
N204F, and a reverse primer
PMP34RNhe. C-terminal deletion mutants were likewise constructed by
PCR, using reverse primers, 204TerR and 186TerR, and a forward primer
HsPMP34F. HA tagging at their C terminus was done as described above.
To construct enhanced green fluorescent protein (GFP)-fusion proteins,
SpeI site was introduced to the initiation codon of
GFP-expression plasmid phGFP105-C1, a generous gift from T. Tsukamoto
and T. Osumi (Himeji Institute of Technology, Himeji, Hyogo, Japan) by
PCR using primers, GFPSpeF and GFPshortR. The resulting PCR fragment
was digested with SpeI and SalI and introduced
into the SpeI-SalI sites of pBluescript SK(
),
termed pBluescript SK(
)GFP. SpeI-SalI fragment
of pBluescript SK(
) GFP and NotI and NheI
fragment of
N125,
N186, 204Ter, or 186Ter, cloned in pGEM/T-easy
vector were introduced into the NotI-SalI site of
pUcD2HygSR
. Chimera constructs for 30/204GFP and 86/204GFP were
generated by PCR, using a forward primer
N30F or
N86F, and a
reverse primer 204TerR. Fusion for 86/273GFP and 125/273GFP were
likewise constructed, using a forward primer
N86F or
N125F, and a
reverse primer 273TerR; GFP tagging was performed as above. Expression
plasmid for 187/204GFP, the fourth loop domain of PMP34 fused to the N
terminus of GFP, was constructed by PCR, using as a template
pUcD2HygSR
·204GFP and as primers
N186F and GFPshortR. For
construction of GFP187/204HA, BglII site was introduced by
PCR on pUcD2HygSR
·204HA using BglII187F and
pUcD2HygSR
reverse (D3R) as forward and reverse primers,
respectively. The BglII and SalI fragments of
187/204HA and NotI-BglII fragment of
pBluescript SK(
) GFP were introduced into the
NotI-SalI site of pUcD2HygSR
.
·HsPMP34.
Cells were stained with rabbit anti-PMP34 C-terminal peptide antibody and guinea pig anti-human catalase antibody, after permeabilization for
5 min using either 25 µg/ml digitonin or for 2 min with 1% Triton
X-100.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of PMP34. A,
intracellular localization of PMP34. HsPMP34-HA was
expressed in wild-type CHO-K1 cells. a and b,
cells were stained with antibodies to HA (a) and Pex14p
(b). Original magnification, ×630; bar, 20 µm.
B, intra-peroxisomal localization. Left,
endogenous PMP34 in rat liver. Rat liver peroxisomes (50 µg) were
analyzed by SDS-PAGE and immunoblot using anti-PMP34 peptide antibody.
Arrowhead indicates ~34-kDa protein band specifically
detected using anti-PMP34 antibody. Asterisk designates a
nonspecific band. Right, membrane (P) and soluble
(S) fractions of CHO-K1 cells expressing PMP34-HA were
prepared from postnuclear supernatant fraction by the sodium carbonate
method (30). Immunoblotting was done using antibodies specific for HA,
AOx, and Pex14p. C, membrane topology of PMP34;
morphological analysis. Transmembrane topology of PMP34 was determined
by the differential cell-permeabilization method. HeLa cells were
transfected with HsPMP34. Cells were fixed and then treated
with 1% Triton X-100 (TX-100; a and
c) or 25 µg/ml digitonin (b and d).
A set of cells were stained for PMP34 using antibodies to PMP34
C-terminal peptide (a and b) and catalase
(c and d). HsPMP34-expressing cells were
indicated by arrowheads (a). Note that C-terminal
part of PMP34, not a matrix enzyme catalase, was detected after both
types of treatments. Original magnification, ×630; bar, 20 µm. D, transmembrane topology of flag-PMP34-HA. CHO-K1
cells transfected with flag-HsPMP34-HA were fixed, then
treated with 1% Triton X-100 (a, c, and
e) or 25 µg/ml digitonin (b, d, and
f). Dual cell staining was done for flag (a and
b) and PTS1 (c and d) using antibodies
to flag and PTS1, respectively. Cells were also stained with anti-HA
antibody (e and f). Note that both N- and
C-terminal parts of PMP34 were detected after both types of treatments.
Original magnification, × 630; bar, 20 µm. E,
sensitivity to protease treatment. PNS fraction of CHO-K1 cells
expressing HsPMP34 was mock-treated (lane 1) or
treated with 4 µg/ml S. aureus V8 protease in the absence
(lane 2) or presence (lane
3) of 1% Triton X-100, for 1.5 h at 25°C. The whole
reaction mixture was analyzed by SDS-PAGE and immunoblot.
Left, immunoblot was done with antibodies to PMP34
(top), Pex14p (middle), and AOx
(bottom). Cells loaded were 2 × 105 (for
the analysis of PMP34) and 7 × 104 (for Pex14p and
AOx), respectively. Solid and open
arrowheads indicate PMP34 and Pex14p; arrow
designates AOx-C component (39). Right, potential cleavage
sites at Glu in PMP34 sequence are indicated at positions 8, 41, 54, 58, 59, 139, 160, 187, 240, 271, and 289. Putative transmembrane
segments are shown in Fig. 2A.
N30HA,
N125HA,
N186HA and
N204HA, lacking the sequence from N
terminus to the first, third, and fourth transmembrane segments, and to
the fourth loop domain, respectively, were HA-tagged at the C terminus.
These truncation mutants were transfected into wild-type CHO-K1 cells
and analyzed for intracellular localization by immunofluorescence
microscopy using anti-HA antibody (Fig. 2B). In the cells
expressing
N30HA, punctate immunofluorescence pattern was observed
and superimposable to that using anti-Pex14p antibody, thus indicating
that
N30HA was localized to peroxisomes (Fig. 2B,
a and b).
N125HA was likewise detected in a
superimposable manner to Pex14p, demonstrating peroxisomal localization
(Fig. 2B, c and d). In the case of
N186HA, punctate pattern was obtained, however, some signal did not
correspond to that obtained from anti-Pex14p antibody (Fig.
2B, e and f, arrowheads).
To confirm the intracellular localization of these two mutants, we also
expressed GFP-tagged
N125 and
N186 and their peroxisomal
localization were analyzed. The full-length PMP34 fused with GFP was
localized to peroxisomes, as seen for PMP34-HA, when expressed in
CHO-K1 cells (data not shown). Punctate signals were observed in the
cells expressing
N125GFP, and colocalized with those noted using
anti-Pex14p antibody (Fig. 2B, g and
h). In the case of
N186GFP, two types of punctate
structures were found, where one type was superimposable with those
seen using anti-Pex14p antibody and the other was not (Fig.
2B, i and j, arrowheads).
N204HA was apparently localized to endoplasmic reticulum-like structures in addition to peroxisomes (Fig. 2B,
k), thereby inferring a decrease in the topogenic activity.
Together, these results suggest that 79-amino acid sequence between the
loop domains 3 (L3) and 4 (L4) is required for localization of PMP34 to
peroxisomes.
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Fig. 2.
Functional and topogenic regions of
PMP34. C-terminally HA-tagged or GFP-fused PMP34 and its variants
were verified for intracellular localization in CHO-K1. A,
constructs of deletion mutants of PMP34. N30HA, PMP34-HA with
deletion of N-terminal residues from 1 to 30; 204HA, HA-tagged PMP34
with residues 1-204; 204GFP, PMP34 comprising residues 1-204 fused
with GFP. Others likewise representing respective constructs were
indicated. Numbers in box represent the positions
of transmembrane segments; L1-L5 designate the
intervening-loop region between two flanking TMs. Peroxisomal targeting
activity of each variant verified (see below) was shown: +, active;
+/
, partially active;
, inactive. B, PMP34 variants
represented in A were expressed in CHO-K1. a and
b,
N30HA; c and d,
N125HA;
e and f,
N186HA; g and
h,
N125GFP; i and j,
N186GFP;
k,
N204HA; l, 186HA; m and
n, 204HA; o and p, 204GFP.
C-terminally HA-tagged PMP34 variants were verified for peroxisomal
localization by immunostaining using mouse (a, c,
e, and m) and rabbit (k and
l) anti-HA antibody and FITC-labeled second antibody, where
peroxisomes were assessed by anti-Pex14p antibody and Texas Red-labeled
second antibody (b, d, f,
h, j, n, and p). PMP34
truncation mutants fused with GFP were verified by GFP fluorescence
(g, i, and o). Arrowheads
indicate PMP34-positive particles, positive in expressed
PMP34-variants, that were absent from Pex14p. Original magnification,
×630; bar, 20 µm. C, transmembrane topology of
GFP fusion proteins,
N125GFP and 204GFP, was determined. CHO-K1
cells expressing
N125GFP (a and b) and 204GFP
(c and d) were fixed, then treated with 25 µg/ml digitonin. Localization and membrane orientation were verified
by GFP fluorescence (a and c) and
immunofluorescence staining of GFP with anti-GFP antibody and Texas
Red-labeled second antibody (b and d).
Bar, 20 µm.
N125GFP and 204GFP, using anti-GFP antibody. Fig. 2C
shows the topology of their C-terminal portion. GFP of the both fusion
proteins was recognized by anti-GFP antibody at 25 µg/ml digitonin,
suggesting that both mutant proteins were integrated into peroxisomal
membranes and exposed the C-terminal part to the cytosol, as wild-type
PMP34. These results demonstrate that the fourth cytosolically faced
hydrophilic loop of 18 amino acids is necessary for targeting of PMP34
to peroxisomes.
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Fig. 3.
Sorting sequence of PMP34. A,
amino acid alignment of putative peroxisome membrane-targeting signal
sequence in PMP34 and mPTS of PMP47. Positively charged amino acids are
underlined. B, site mutation of the loop region
between TM4 and TM5 of PMP34. C, subcellular localization of
PMP34 variants mutated in the loop region. Each construct was expressed
in CHO-K1 cells and detected by GFP fluorescence. a, 6A204;
b, 4A204; c, 3A204; d, 2A204;
e, A204. Bar, 20 µm.
View larger version (62K):
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Fig. 4.
The fourth basic loop sequence is not
sufficient for peroxisomal localization. A, constructs
of the loop region fused with GFP. A partial PMP34 consisting of amino
acid residues at 187-204 was fused to GFP. B, intracellular
localization of the loop-GFP fusion protein. Each construct was
expressed in CHO-K1 cells and detected by fluorescence as in Fig.
3C. a, 187/204GFP; b, GFP187/204HA.
Bar, 20 µm.
N125GFP which possess four and three
transmembrane segments, respectively, were localized to peroxisomes as
efficiently as wild-type PMP34, suggesting that the region(s) required
for integration to peroxisomal membranes is in both N- and C-terminal domains (see above).
N125GFP with three TMs containing the fourth TM, was localized to peroxisomes, while
N186GFP with two TMs, but
lacking the fourth TM, was localized to not only peroxisomes but also
another organelles. These results suggest two possibilities for
integration into peroxisomal membrane. One is that fourth TM is
important for integration into peroxisomal membranes. The other is that
at least three TMs are required. To address this issue, two mutants,
86/204GFP and 125/273GFP, containing the fourth TM plus one TM, were
constructed (Fig. 5A). Their
intracellular localization is shown in Fig. 5B. Both
86/204GFP and 125/273GFP were not localized to peroxisomes (Fig.
5B, a and e). It is of interest to
note that 86/204GFP was instead transported to mitochondria, as
verified by staining using anti-malate dehydrogenase antibody (Fig.
5B, b). These results suggest that at least three
TMs, not the fourth TM, are required for integration into peroxisomal
membranes.
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Fig. 5.
Coordinated function of the membrane
targeting sequence and transmembrane segments. A,
constructs of the loop region and transmembrane segments (loop plus TM)
fused with GFP. B, intracellular localization of the (loop
plus TM)-GFP fusion protein. a and b, 86/204GFP;
c and d, 30/204GFP; e, 125/273GFP;
f, 86/273GFP. Each construct was expressed in CHO-K1 cells
and detected by GFP fluorescence (a, c,
e, and f). Cells expressing 86/204GFP were also
stained using anti-malate dehydrogenase antibody and Texas Red-labeled
second antibody (b); peroxisomes in 30/204GFP-expressing
cells were assessed by anti-Pex14p antibody (d).
Bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix structure (data not shown). Moreover, Ala is not a helix breaker. Therefore, it is most likely that the positively charged
loop works as mPTS.
N125GFP, were specifically localized to
peroxisomes as efficiently as the full-length PMP34-GFP, although 86/273GFP and 86/230GFP, both despite with three TMs 3-5, were not
localized to peroxisomes; 3) 30/204GFP with deletion of TM1 of TMs
1-4, or
N186GFP likewise deleted in TM4 of TMs 4-6 was significantly reduced in the level of peroxisomal localization. Thus,
we infer that three TMs are necessary to integrate into peroxisome
membrane. This conclusion was confirmed by Ala substitution mutants.
Since Ala substitution mutants were integrated into peroxisomal membrane in the same manner as wild-type PMP34, although their targeting activity is weak, the implication is that integration of
PMP34 is dependent on its transmembrane segment but does not depend on
the fourth loop domain. It is of interest to note that the fourth basic
loop domain fused to GFP was transported to mitochondria, presumably
recognized as a mitochondrial targeting signal. Hence, three TMs are
more likely to play a role in allowing the basic loop to be readily
recognized by a putative mPTS recognition factor (see Fig.
6, X) and not by a
mitochondrial signal receptor, as was seen in the case of 204GFP and
N125GFP specifically localized to peroxisomes.
View larger version (20K):
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Fig. 6.
A schematic view of PMP34 topogenesis.
After synthesis on cytoplasmic free polyribosomes, PMP34 is recognized
by a soluble factor such as putative "mPTS receptor" (designated by
X) at the loop region between TM4 and TM5, transported to
the surface of peroxisome membrane, imported by a "membrane protein
import receptor," then finally localized to the membrane by vectorial
translocation. At least three TMs are also responsible for the
targeting and/or integration.
Several peroxisomal proteins have been suggested to be glycosylated, including N-glycosylated Pex2p and Pex16p of Y. lipolytica (11). We investigated this issue extensively using mammalian cells. Mutation of two potential N-glycosylation sites, including N167GT to DGT or N241RT to DRT did not alter the mobility of PMP34 in SDS-PAGE (data not shown), suggesting that PMP34 is not N-glycosylated. Moreover, we observed no staining of endoplasmic reticulum, using anti-PMP34 antibody, in CHO-K1 cell overexpressing PMP34 (data not shown). These results imply that endoplasmic reticulum is less likely to be involved in biogenesis of PMP34.
Given the findings described here, we propose a hypothetical model of
the topogenesis of PMP34 (Fig. 6). After the synthesis on cytoplasmic
polysomes, PMP34 is transported to peroxisomes in an
mPTS-dependent manner, and inserted into membranes with the
aid of at least three hydrophobic TMs, such as TMs 1-3 and 4-6. Each
of three TMs may form a targeting-competent conformation, a module-like
structure, that enables PMP34 to be recognized by a cytosolic factor
(Fig. 6, X) and/or to be readily integrated. The complex
then binds to a putative membrane protein import receptor, and PMP34 is
finally localized into peroxisome membranes. The lower hydrophobicity
of the TMs of PMP34, as compared with other PMPs, such as Pex3p, may
require three TMs to maintain such conformation. Similar mechanisms are
involved in topogenesis of the ADP/ATP carrier in mitochondrial inner
membrane (37). This topogenesis mechanism is compatible with two
previous observations on the in vitro import of peroxisomal
membrane proteins, PMP22 (9) and PMP70 (10). The peroxin Pex19p is a
partially farnesylated, acidic peroxin responsible for peroxisome
biogenesis disorders of complementation group J (38). Pex19p shows a
dual intracellular localization, on peroxisome membranes and in the
cytosol. It is noteworthy that Pex19p was very recently reported to
weakly interact with PMP34 (36). The region containing the basic loop
of PMP34 may mediate binding to Pex19p. Other cytosolic factors may
also be involved in the transport of PMP34.
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ACKNOWLEDGEMENTS |
---|
We thank R. Tanaka for help in preparing figures and the other members of the Fujiki laboratory for discussion. We also thank N. Thomas for comments.
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FOOTNOTES |
---|
* This work was supported in part by a CREST grant (to Y. F.) from the Japan Science and Technology Corporation, and by Grants-in-aid for Scientific Research 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, 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, December 19, 2000, DOI 10.1074/jbc.M003304200
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ABBREVIATIONS |
---|
The abbreviations used are: PTS, peroxisome targeting signal; AOx, acyl-CoA oxidase; FITC, fluorescein isothiocyanate; GFP, enhanced green fluorescent protein; HA, influenza virus hemagglutinin; mPTS, peroxisome targeting signal for membrane protein; PAGE, polyacrylamide gel electrophoresis; PMP47, 47-kDa peroxisomal integral membrane protein; PMP34, 34-kDa PMP; TM, transmembrane segment; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PNS, postnuclear supernatant.
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