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INTRODUCTION |
The regulation of organelle abundance involves a complex interplay
of organelle synthesis and destruction. In the case of peroxisomes,
little is known about how cells control these competing processes.
Peroxisome destruction appears to occur by a specialized form of
autophagy (pexophagy) which requires most APG/CVT
genes. In addition to the APG/CVT genes
that are involved in general autophagy, some specific factors are
required for pexophagy (1). Peroxisome formation appears to be even
more complex. Many lines of evidence suggest that peroxisomes are
formed mainly from preexisting peroxisomes, either by budding or
fission, and this may be the predominant mechanism for peroxisome
formation (2, 3). However, peroxisome synthesis has also been observed
in the absence of preexisting peroxisomes (4, 5), indicating that there
may be two parallel pathways for peroxisome formation, one from
preexisting peroxisome and another de novo pathway for
peroxisome formation (4, 6).
Of the many PEX1
genes and products (peroxins) required for peroxisome biogenesis, only
PEX11 has been shown to have a conserved role in peroxisome
division (7-13). The overexpression of PEX11 promotes peroxisome
elongation and subsequent division, whereas loss of PEX11 results in
reduced peroxisome abundance (7, 8, 12, 14). There is also evidence for
metabolic control of peroxisome division (6, 15, 16), although PEX11
proteins can induce peroxisome proliferation independently of
peroxisome metabolism (17). These results indicate that PEX11 proteins
may play a positive role in the division process. In human cells,
PEX11-mediated peroxisome division involves three steps: first, the
import of PEX11 into the peroxisome membrane; second, the elongation of peroxisomes and the segregation of PEX11 proteins away from other peroxisomal membrane proteins, forming PEX11-enriched patches; and
third, the division of elongated peroxisome tubules into multiple, small peroxisome vesicles (12, 17). However, the biochemical activities
of PEX11 proteins remain obscure, and there is as yet no mechanistic
model for PEX11-induced peroxisome division. There may also be
species-specific differences in PEX11 function because mammalian cells
express at least three distinct PEX11 genes, whereas Saccharomyces cerevisiae has a single PEX11 gene
(18).
Recently, Hoepfner et al. (19) reported that the yeast
dynamin-related GTPase, Vps1p, is also involved in peroxisome division. Yeast vps1 null mutants contain only one or two large
peroxisomes and cannot increase peroxisome abundance under conditions
that normally induce PEX11 expression and promote peroxisome
division (19). To test whether a similar process exists in mammalian cells and to determine further whether this is related to
PEX11-mediated peroxisome division, we studied the role of human DLP1
in peroxisome biogenesis. DLP1 is the human gene most
similar to two of three dynamin-like proteins found in yeast, Vps1p and
Dnm1p. DLP1 is also known to regulate the dynamics of mitochondria and
the endoplasmic reticulum (20, 21). We report here that a reduction in
DLP1 activity inhibits peroxisome division and reduces peroxisome
abundance. Furthermore, we provide evidence that PEX11 overexpression
recruits DLP1 to peroxisomes, providing a potential mechanism for
PEX11-induced peroxisome division.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The plasmids pcDNA3-PEX11
myc
(12), pcDNA3- PMP34myc (22, 23),
pPGK1-GFP/PTS1, and
pRS425/GAL1-PEX11 (17) have been described. The plasmids
expressing COOH-terminal 3×HA-tagged PEX11
, PEX11
, PEX11
, and
PMP34 were created by inserting above four full-length genes into a
modified pcDNA3-3×HA vector (4). The plasmid
pcDNA3-PMP34VSVG was cloned by inserting full-length
PMP34 gene upstream (in-frame with) of a VSVG epitope in a
modified pcDNA3-VSVG vector (4).
The human DLP1 gene was cloned by reverse transcription-PCR
against the total RNA of human fibroblasts with a primer (DLP1-RT: 5'-CCACATGAGCAGATTCATACC-3') designed according to the 3'-untranslated region of human DLP1 cDNA for reverse transcription and
two DLP1-specific primers (DLP1-5',
5'-CCCCTCGAGGGAGGCGCTAATTCCTGTCATAAAC-3', and DLP1-3',
5'-CACCGCGGCCGCTCACCAAAGATGAGTCTCCCGG-3') for PCR. The resulting
PCR products were then digested with XhoI-NotI
and cloned downstream of (in-frame with) a c-myc epitope in a modified
pcDNA3-Nmyc vector. Plasmids from various colonies were
sequenced, and three isoforms of DLP1 (DLP1 a
form, b form, and c form, see Fig. 9) have been
identified in fibroblasts. The full-length DLP1 gene was
cloned by inserting the deleted nucleotides into the DLP1c by PCR. The dominant negative forms of DLP1
(DLP1S39N and DLP1T59A) were created by PCR
site-directed mutagenesis using the oligonucleotides DLP1-5' and
DLP1S39N-3',
5'-GAAGCAGGTCCCTCCCCACCAGGCTTTCTAGCACTGAGTTCTTTC-3', and DLP1-5'
and DLP1T59A-3', 5'-GAGAGGTCTCCGGGCGACAATTCC-3', respectively. The resulting PCR fragments were then digested with
XhoI-PpuMI and XhoI-BsaI,
respectively, and used to replace their corresponding sequence in
pcDNA3- NmycDLP1.
Antibodies--
Two affinity-purified rabbit anti-DLP1 peptide
antibodies anti-DLP1MID and anti-DLP1N were provided by Dr. McNiven
(Mayo Clinic and Foundation, Rochester, MN) (20). Antiserum against the
COOH-terminal 16 amino acids of DLP1 (anti-DLP1C) was generated by
injecting rabbits with ovalbumin (Pierce) coupled peptide
NH2-CKQGASQIIAEIRETHLW-COOH. Sheep antibodies against PMP70
and rabbit antibodies against PEX13 have been described (4). The
monoclonal antibodies to the c-myc epitope were obtained from the
tissue culture supernatant of the hybridoma 1-9E10 (24). Polyclonal
antibodies to the c-myc epitope and anti-HA monoclonal antibodies
coupled to agarose beads were obtained from the Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-HA monoclonal antibodies were
obtained from Roche Applied Science. Secondary antibodies specific for
sheep, rabbit, and mouse antibodies were obtained from commercial sources.
Cell Lines, Transfection, and Immunofluorescence--
An
immortalized wild type human skin fibroblast GM5756-Ti and the wild
type and PEX11
-deficient mouse embryonic fibroblasts (MEFs) (14) were cultured under standard conditions (25). All
transfections were performed by electroporation (26). For immunofluorescence, cells were fixed in 3% formaldehyde in Dulbecco's modified phosphate-buffered saline, pH 7.1 (DPBS, Invitrogen), for 20 min and then permeabilized in 25 µg/ml digitonin in DPBS for 5 min.
Cells were then incubated with primary antibodies for 30 min, washed
extensively in DPBS, incubated with fluorescently labeled secondary
antibodies for 10 min, washed extensively in DPBS, and mounted on
slides. Peroxisome abundance in fibroblasts was determined as described
previously (17).
RNA Interference--
To knock down the expression of DLP1 in
fibroblasts by RNA interference, two pairs of DLP1
gene-specific 21-nucleotide small interfering RNA (siRNA) (sense
strand: DLP1·siRNA1, 5'-UAGCUGCGGTGAACCCGUGdTdT-3'; and
DLP1·siRNA2, 5'-CCAACCACAGGCAACUGGAdTdT-3') and one pair of control
PXR2 siRNA (sense strand: PXR2·siRNA,
5'-GCAGGGAAAAGGCUCUAGGdTdT-3') were synthesized by Dharmacon
Research (Lafayette, CO) and annealed according to the manufacturer's
guidelines. 50 µl of 20 µM siRNA duplexes was then
transfected into fibroblasts from one confluent T-75 flask by
electroporation (26), and the DLP1 protein level and peroxisome
morphology and abundance were analyzed at various time points by
immunoblot and indirect immunofluorescence, respectively. To examine
the peroxisome proliferating activity of PEX11
in siRNA
duplex-treated cells, fibroblasts were first transfected with
DLP1 or PXR2 siRNA duplexes and cultured for
24 h, then transfected with PEX11
expression vector
pcDNA3-PEX11
myc together with another dose of siRNA
duplexes. Cells were then cultured for an additional 48 h before
analyzing the peroxisome abundance and morphology.
Immunoprecipitation--
To study the association of DLP1 with
peroxisomes, fibroblasts expressing the COOH-terminal 3×HA-tagged or
VSVG-tagged PMP34 in combination with mycDLP1aT59A were harvested by
scraping in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and CompleteTM protease inhibitor mixture
(Roche Applied Science) and lysed by passing through a 22-gauge syringe
needle five times. Lysates were then subjected to centrifugation at
25,000 × g for 15 min to pellet large organelles. The
resulting supernatant was diluted in TBS buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and
CompleteTM protease inhibitor mixture). Immunoprecipitation was
initiated by adding 15 µl of a 50% slurry of anti-HA monoclonal antibodies coupled to agarose beads into 500 µl of the above
supernatant together with 1% bovine serum albumin. The mixture was
then incubated with mixing at 4 °C for 2 h, and anti-HA beads
were pelleted by brief centrifugation, washed five times with 1 ml of
TBS, and resuspended in SDS-sample buffer. The supernatant and
corresponding immunoprecipitate sample were then separated by SDS-PAGE,
transferred to polyvinylidene difluoride membranes, and probed with
anti-myc polyclonal antibodies.
To study the possible interaction between PEX11 proteins as well as
that between PEX11 and DLP1, fibroblasts expressing the appropriate
proteins were lysed by incubation with mixing in TBS buffer plus 1%
Triton X-100 or 0.2% digitonin at 4 °C for 1 h. Lysates were
then subjected to centrifugation at 100,000 × g for 30 min to pellet cellular membranes. The resulting supernatant was diluted
into TBS buffer with detergent and 1% bovine serum albumin.
Immunoprecipitation was carried out by adding either 15 µl of a 50%
slurry of anti-HA beads or 20 µl of a 50% slurry of protein
A-agarose (Sigma) plus 10 µl of 200 µg/ml anti-myc polyclonal
antibodies and incubating with mixing at 4 °C for 2 h. The
beads were then pelleted, washed extensively 3 × 1 ml with TBS
buffer with detergent, 3 × 1 ml with high salt TBS buffer (50 mM Tris-HCl, pH 7.5, 350 mM NaCl, 1 mM EDTA, and CompleteTM protease inhibitor mixture with
detergent), and 3 × 1 ml with TBS buffer. The beads were
resuspended in SDS-sample buffer, separated together with
corresponding supernatant by SDS-PAGE, and probed with either anti-myc
polyclonal antibodies or anti-HA monoclonal antibodies.
Peroxisome Purification by Nycodenz Gradient--
To study the
association of DLP1 with peroxisomes, fibroblasts expressing PMP34myc
or PEX11
myc were harvested by trypsin digestion, washed with PBS
buffer once and with homogenization buffer (0.25 M sucrose,
1 mM Tris-HCl, pH 7.5, and 1 mM EDTA) once.
Then the cells were resuspended in 1.2 ml of homogenization buffer with
CompleteTM protease inhibitor mixture and homogenized by five passes
through a precision ball-bearing homogenizer. Homogenates were
centrifuged for 30 s at 1,500 × g to yield a
postnuclear supernatant. The resulting postnuclear supernatant was
loaded on top of 10.0 ml of linear 15-42% nycodenz gradient with a
cushion of 1.5 ml of maxidenz and centrifuged at 4 °C for 25 min at
137,500 × g. 0.75-ml fractions were collected and
analyzed for catalase activity and succinate dehydrogenase activity as
described (27).
Yeast Strains and Culture Conditions--
Yeast strains used in
this study were based on S. cerevisiae strain BY4733
(MATa, his3
200, leu2
0, met15
0, trp1
63,
ura3
0). The strain XLY1 (pex11
) was generated as
described previously (17). The XLY3 strain (vps1
) was
generated by one-step PCR-mediated disruption of the VPS1
gene in BY4733 using HIS3 as the selectable maker (28). The
strain XLY4 (pex11
, vps1
) was generated from XLY1 by
one-step PCR-mediated disruption of the VPS1 gene using HIS3 as the selectable maker. Plasmid transformations were
performed using the LiOAc procedure (29). All strain were cultured in minimal S medium (0.17% yeast nitrogen base w/o ammonium sulfate (Sigma), 0.5% ammonium sulfate) with 2% glucose, 1% galactose, or
0.2% oleic acid or 0.02% Tween 40 as carbon source. Media were supplemented with amino acids, uracil, and adenine as required (29).
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RESULTS |
Reduced DLP1 Levels Cause Reduced Peroxisome Abundance--
The
observation that yeast Vps1p participates in peroxisome membrane
division suggested that the human ortholog of Vps1p could have a
similar role. However, the human DLP1 gene product, which is
the human protein that shows the greatest sequence similarity with
S. cerevisiae Vps1p, shares even greater similarity with S. cerevisiae Dnm1p, a yeast protein that regulates
mitochondrial division and endosome dynamics but has no discernible
role in peroxisome division (19, 30). Furthermore, human DLP1 has been
shown previously to regulate mitochondrion division and endoplasmic reticulum dynamics (20, 21), indicating that it might share more
functional similarity with Dnm1p than with Vps1p. Therefore, it was
essential to empirically determine whether human DLP1 has a role in
peroxisome biogenesis.
We first attempted to reduce DLP1 mRNA and DLP1 protein
levels using small interfering RNA (siRNA) specific for the
DLP1 message. Two pairs of DLP1 siRNA duplexes
(DLP1· siRNA1 and DLP1·siRNA2) and a control siRNA of a
nonperoxisomal gene PXR2 (PXR2·siRNA) were designed
according to Elbashir et al. (31). After annealing, they
were transfected into an immortalized human skin fibroblast cell line
(5756-Ti) by electroporation. Cells were then incubated at 37 °C and
processed for immunoblot and indirect immunofluorescence analyses at
various times after transfection. DLP1 is known to have
multiple isoforms that are generated by alternative splicing of the
initial DLP1 transcript (20, 33). Our immunoblot analysis indicated that at least two different isoforms were present in fibroblasts (Fig. 1A). The
immunoblot results also confirmed that both DLP1·siRNA
duplexes were able to reduce DLP1 protein level, and the maximal
reduction (to 30-40% of normal level) was reached by day 4 after
transfection (Fig. 1A). Control PXR2·siRNA, on the other
hand, had no effect on the DLP1 protein level (Fig. 1A).
Given that the transfection efficiency is typically about 80-90% for
the siRNA duplex in these cells, DLP1 protein levels are likely to be
even lower than 30% of normal level in those cells that took up the
siRNA to DLP1. As expected, peroxisome abundance in
untransfected cells (141 ± 46 pps) was similar to that of cells
transfected with the control siRNA (138 ± 46 pps) (Fig.
1B). However, cells transfected with the DLP1·siRNA
contained fewer peroxisomes (58 ± 23 pps). Furthermore, the
morphology of peroxisomes in these cells was quite atypical, with
peroxisomes in these cells having a long, tubular morphology (Fig. 1,
E and F) compared with the small, punctuate
morphology of normal peroxisomes (Fig. 1, C and
D). Control experiments with antibodies to the peroxisomal
matrix enzyme catalase indicated that peroxisomal protein import was
unaffected even in the cells with the most severe reductions of
peroxisome abundance (data not shown).

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Fig. 1.
RNA interference of DLP1 results in reduced
peroxisome abundance. Wild type human skin fibroblasts 5756-Ti
were transfected with PXR2·siRNA or DLP1·siRNA1 duplexes, and cells
were then harvested for immunoblot (A) or processed for
double indirect immunofluorescence (B-F) at various times
after transfection. A, whole cell lysates from different
days were separated by SDS-PAGE and probed with DLP1 antibodies
(DLP1MID) and PEX13 antibodies. B, peroxisome abundance in
5756-Ti cells, 5756-Ti cells transfected with PXR2·siRNA, and 5756-Ti
cells transfected with DLP1·siRNA1. C-F, representative
cells treated with PXR2·siRNA (C and D) and
DLP1· siRNA1 (E and F). Cells were processed
at 48 h after transfection for immunofluorescence with antibodies
to PMP70 and catalase (not shown). Samples were examined by confocal
fluorescence microscope, and the number of peroxisomes present in at
least 30 randomly selected cells was counted. Results in B
are presented as the average peroxisome abundance ± 1 S.D.
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Dominant Negative DLP1 Mutants Reduce Peroxisome Abundance--
As
an independent test of DLP1 involvement in peroxisome division, we
expressed wild type and dominant negative DLP1 mutants in
human fibroblasts and assessed their effects on peroxisome abundance.
DLP1 shares a high degree of sequence similarity with dynamin,
especially in the amino-terminal tripartite GTP binding motif. Previous
studies have identified two dominant negative dynamin genetic mutants
that exhibit reduced GTP binding affinity (S45N) and reduced GTP
hydrolysis activity (T65A), respectively (32). We therefore generated
the corresponding mutations in the DLP1 cDNA (S39N and
T59A, respectively). Amino-terminally myc-tagged forms of wild type
DLP1a (one of several DLP1 isoforms generated by alternative splicing
(20, 33)) and each of these single substitution mutants of DLP1a were
transfected into human fibroblasts. Four days after transfection each
cell population was processed for indirect immunofluorescence using
antibodies specific for the myc epitope tag and PMP70, an integral
peroxisomal membrane protein marker.
Peroxisome abundance in cells expressing wild type DLP1a (123 ± 46 pps) was similar to that in untransfected cells (141 ± 46 pps)
(Fig. 2A, compare first
two bars, p = 0.194, Student's t
test), and the overexpressed, myc-tagged form of DLP1a appeared to be
largely cytosolic (Fig. 2, B and C). On the other
hand, the peroxisome abundance was reduced (71 ± 37 pps) (Fig.
2A, p < 0.001, Student's t
test) in cells expressing the DLP1aS39N mutant, which has a reduced
affinity for GTP (32). Similar to the DLP1 RNA interference cells, some
of the DLP1aS39N-expressing cells have only a couple of giant,
reticular peroxisomes (Fig. 2, D and E). This
DLP1a mutant appeared to be primarily cytosolic like wild type protein
(Fig. 2D). Overexpression of the DLP1aT59A mutant also
reduced peroxisome abundance in these cells (83 ± 26 pps) (Fig.
2A, p = 0.011, Student's t
test). However, this DLP1 mutant appeared to be associated principally
with small punctuate structures, few if any of which appeared to be
peroxisomes (Fig. 2, F and G).

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Fig. 2.
Overexpression of dominant negative mutants
of DLP1 reduces peroxisome abundance.
A, peroxisome abundance in 5756-Ti cells and 5756-Ti cells
overexpressing mycDLP1a, mycDLP1aS39N, or mycDLP1aT59A.
B-G, representative cells transfected with
pcDNA3- mycDLP1a (B and C),
pcDNA3-mycDLP1aS39N (D and E), or
pcDNA3- mycDLP1aT59A (F and G).
5756-Ti cells were transfected with appropriate plasmids. 4 days after
transfection, cells were processed for indirect immunofluorescence with
antibodies to the myc epitope (B, D, and
F) and PMP70 (C, E, and G).
Samples were examined by confocal fluorescence microscope, and the
number of peroxisomes present in at least 30 randomly selected cells
was counted. Results in B are presented as the average
peroxisome abundance ± 1 S.D.
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A Subpopulation of DLP1 Colocalizes with PEX11-induced, Elongated
Peroxisomes--
Previous studies of dynamin and DLP1 have indicated
that these large GTPases may act as "pinchases," promoting
vesicle budding and organelle fission by contracting membrane tubules
(34, 35). If applied to peroxisome division, this model would predict
that DLP1 physically associates with peroxisomes during their division.
To test for DLP1 association of peroxisomes, we generated rabbit
antibodies (DLP1C) against the COOH-terminal 16 amino acids of DLP1 and
used them for immunofluorescence analysis. Under normal conditions, the
endogenous DLP1 proteins localize in small cytoplasmic vesicles in
fibroblasts (Fig. 3, B and
E). Although the majority of DLP1-positive vesicles were
nonperoxisomal, a small subpopulation of DLP1-positive vesicles also
overlapped with the peroxisomal membrane marker protein PMP70 (Fig.
3C, indicated by arrowheads).

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Fig. 3.
A subpopulation of DLP1 colocalizes
with peroxisomes. 5756-Ti cells (A, B, and
C) and 5756-Ti cells expressing PEX11 myc (D,
E, and F) were processed for double indirect
immunofluorescence for DLP1 (green) and peroxisomal membrane
marker PMP70 (red) (C) or myc epitope
(red) (F). Note that although the majority of
DLP1-positive structures are cytoplasmic, a subpopulation of them are
associated with peroxisomes (arrowheads in C
inset), especially along the elongated peroxisomes in
PEX11 myc-expressed cells (arrowheads in F
inset).
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To examine the association of DLP1 with peroxisomes during the division
process, we took advantage of the fact that overexpression of PEX11
proteins induces peroxisome division. We overexpressed myc-tagged
PEX11
protein in fibroblasts and then used antibodies specific for
DLP1 (DLP1C) and confocal immunofluorescence microscopy to detect DLP1
localization. We also used the antibodies to PMP70 and the myc epitope
to label peroxisomes. Under these conditions, more DLP1 was colocalized
with peroxisomes, and some DLP1 signals were aligned along the
elongated peroxisome tubules prior to their division (Fig.
3F, indicated by arrowheads). Given that
PEX11-mediated peroxisome division appears to involve multiple stages,
particularly peroxisome elongation and subsequent division (12, 17),
these observations suggest that DLP1 associates with peroxisomes prior to or during the division process. However, this interaction is likely
to be transient because the majority of DLP1 does not overlap with peroxisomes.
We next asked whether we could collect biochemical evidence for
peroxisome-associated DLP1. We transfected 5756-Ti cells with a plasmid
designed to express mycDLP1T59A, a dominant negative mutant predicted
to lack GTPase activity (a similar mutant of dynamin displays increased
association with target membrane (32)), and a plasmid expressing
HA-tagged PMP34. The lysates were prepared from transfected cells, and
then the peroxisomal membranes were immunopurified with anti-HA
monoclonal antibodies. We used anti-myc antibodies to detect myc-tagged
DLP1T59A and found that DLP1T59A was coimmunopurified with peroxisome
membranes (Fig. 4Aa). As a
control for the nonspecific binding of peroxisomal membranes to anti-HA
beads, parallel experiments were carried out with cells cotransfected
with plasmids expressing VSVG-tagged PMP34 and myc-tagged DLP1T59A.
Like PMP34-myc, PMP34-VSVG was also targeted into peroxisomes and had
no effect on peroxisome abundance in fibroblasts (not shown). This
control revealed that coimmunoprecipitation of DLP1 with peroxisomes
was specific, with a signal of ~2-fold over background (Fig.
4Ab). This experiment was repeated several times, and each trial yielded essentially the same results. Furthermore, similar results were obtained when other peroxisomal membrane proteins were
used for immunopurification of peroxisomal membranes (data not
shown).

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Fig. 4.
A small population of DLP1 is associated with
peroxisomes. A, 5756-Ti cells expressing PMP34-3×HA
and mycDLP1aT59A (a) or PMP34-VSVG and mycDLP1aT59A
(b) were lysed by thorough mixing in hypotonic buffer, and
the lysates were cleared by centrifugation at 25,000 × g for 15 min. The resulting supernatant was processed for
immunoprecipitation using anti-HA monoclonal antibodies. The
supernatant and corresponding immunoprecipitate sample were then
separated by SDS-PAGE and probed with anti-myc polyclonal antibodies
(DLP1T59A). DLP1as, amount of DLP1aT59A associated with
peroxisomes; DLP1in, amount of DLP1aT59A in input
supernatant. Please note that although immunoprecipitates of
a and b were loaded equally, and the supernatants
of a and b were also loaded equally, the
immunoprecipitate (IP) and corresponding supernatant for
each sample were not loaded equally (supernatant is only ~4% of the
immunoprecipitate sample) because of the detection limit of the
immunoblot. Therefore, the relative amount (DLP1as/DLP1in) can only be
used for the relative comparison between samples a and
b. B, fibroblasts expressing PMP34myc
(c) or PEX11 myc (d) were homogenized and
centrifuged. The resulting postnuclear supernatant was fractionated
into peroxisome, mitochondrion, and cytosol fractions. The peak of the
peroxisome fraction was then separated by SDS-PAGE and probed with
anti-DLP1MID and anti-PEX13 antibodies. C, 5756-Ti cells
expressing PMP34-3×HA and mycDLP1a (e) or PEX11 -3×HA
and mycDLP1a (f) were lysed in hypotonic buffer and cleared
by centrifugation at 25,000 × g for 15 min. The
resulting supernatant was processed for immunoprecipitation using
anti-HA monoclonal antibodies. The supernatant and corresponding
immunoprecipitate sample were then separated by SDS-PAGE and probed
with anti-myc polyclonal antibodies (DLP1a).
DLP1as, the amount of DLP1a associated with peroxisomes;
DLP1in, the amount of DLP1a in the input supernatant. The
relative amount (DLP1as/DLP1in) is defined the same as in A.
The densitometry results of A-C show that DLP1 is
associated with peroxisomes (a and b) and that
peroxisome-associated DLP1 is increased in PEX11 -expressing cells
(c-f).
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As independent measurements of DLP1 association with peroxisomes, we
used nycodenz gradient centrifugation to purify peroxisomes from human
fibroblasts that were transfected with PMP34myc (Fig. 4Bc) or PEX11
myc (Fig. 4Bd). Peak
peroxisome fractions were then separated by SDS-PAGE and blotted with
anti-DLP1MID, an antibody that recognizes a DLP1 peptide from the
middle region of this protein (20), and antibodies specific for PEX13,
a peroxisomal membrane protein marker (Fig. 4B). The
relative level of peroxisome-associated DLP1 was ~2-fold higher in
cells expressing PEX11
compared with those expressing PMP34,
suggesting that DLP1 is recruited to peroxisomes during division. A
PEX11
-mediated increase of peroxisome-associated DLP1 was also
detected with immunopurification of peroxisome membranes (Fig.
4C). We coexpressed mycDLP1a together with PMP34-3×HA (Fig. 4Ce) or PEX11
-3×HA (Fig. 4Cf) in 5756-Ti
cells, then lysed the cells and purified peroxisome membranes with
anti-HA monoclonal antibodies. The copurified mycDLP1 were detected by
anti-myc antibodies. Again, the relative level of peroxisome-associated
DLP1 was about 1.6-fold higher in cells expressing PEX11
than in
control cells (Fig. 4C).
Inhibition of DLP1 Blocks PEX11-mediated Peroxisome
Proliferation--
The apparent recruitment of DLP1 to peroxisomes by
PEX11
raises the possibility that DLP1 is required for
PEX11-medidated peroxisome proliferation. To address this issue we
reduced DLP1 in human skin fibroblasts with the DLP1·siRNA, then
transfected these cells with the PEX11
myc expression vector to
induce peroxisome division. Overexpression of PEX11
myc induced a
4-fold increase in peroxisome abundance in untransfected cells or cells
pretransfected with the control siRNA (PXR2·siRNA) for 24 h
(Fig. 5A). In contrast, PEX11
overexpression was unable to induce peroxisome proliferation in cells
pretransfected with DLP1·siRNA for 24 h (Fig. 5A). In the cells pretransfected with DLP1·siRNA, PEX11
protein still stimulated peroxisome elongation but did not induce peroxisome division
(Fig. 5D). Interestingly, PEX11
overexpression still led
to the segregation of peroxisome membrane into subregions that were
either rich in PEX11
proteins or were enriched in other peroxisomal
membrane proteins (Fig. 5, compare D and E, the
merged image is shown in the inset of D). Similar
results were also observed when dominant negative DLP1 mutants were
expressed (data not shown).

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Fig. 5.
Inhibition of DLP1 blocks the
PEX11 -mediated peroxisome
proliferation. A, peroxisome abundance in 5756-Ti
cells, 5756-Ti cells overexpressing PEX11 myc, 5756-Ti cells treated
with PXR2·siRNA/PEX11 myc, and 5756-Ti cells treated with
DLP1·siRNA1/PEX11 myc. 5756-Ti cells were transfected with
the control PXR2·siRNA or DLP1·siRNA1, incubated for 1 day,
transfected with the PEX11 myc expression vector and another dose of
siRNA duplexes. After an additional 2 days cells were processed for
immunofluorescence microscopy using antibodies to the myc epitope tag
(B and D) and PMP70 (C and
E). B-E, representative cells transfected with
PXR2·siRNA and PEX11 myc expression vector (B and
C), and DLP1·siRNA1 and PEX11 myc expression vector
(D and E). The inset in D
is the merged image of the boxed area in D and
E, indicating the segregation of PEX11 myc
(green) from PMP70 (red) along the elongated
peroxisomes in DLP1·siRNA-treated cells. Bar, 1 µm.
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To assess further the connection between DLP1 and PEX11-mediated
peroxisome division, we expressed the myc-tagged wild type DLP1a or
dominant negative mutant DLP1aS39N into wild type and PEX11
/
mouse embryonic fibroblasts. The
peroxisomes in the transfected cells were then examined with
immunofluorescence microscopy using antibodies for the myc epitope
(Fig. 6, A, C,
E, and G) or PMP70 (Fig. 6, B,
D, F, and H). In wild type cells, the
expression of DLP1a had no significant effect on either peroxisome
abundance or morphology (Fig. 6, A and B).
Expression of DLP1aS39N, on the other hand, caused reduction of
peroxisome abundance and peroxisome elongation (Fig. 6, C
and D). As described previously (14), peroxisomes of
PEX11
/
MEFs were reduced in abundance
by about 50% (Fig. 6, E and F). Expression
of DLP1aS39N in PEX11
/
MEFs exacerbated
this phenotype (Fig. 6, G and H).

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Fig. 6.
Dominant negative mutant of DLP1 (DLP1S39N)
reduces peroxisome abundance and induces tubular peroxisomes in
PEX11 /
MEFs. Wild type (WT) and
PEX11 / MEFs were transfected with
pcDNA3-mycDLP1a or pcDNA3-mycDLP1aS39N
and processed for indirect immunofluorescence with antibodies for myc
epitope (A, C, E, and G)
and PMP70 (B, D, F, and H).
Note that in wild type MEFs, the expression of DLP1aS39N induced fewer
and elongated peroxisomes (D). In
PEX11 / MEFs, expression of DLP1aS39N
induced more elongated peroxisomes than PEX11 deficiency
alone, and the peroxisome abundance decreased further.
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Yeast Vps1p Is Required for PEX11-mediated Peroxisome
Proliferation--
It was reported previously by Hoepfner et
al. (19) that peroxisome abundance was reduced significantly in a
vps1 null mutant of yeast. To improve our understanding of
the roles of Vps1p and Pex11p in peroxisome division, we examined the
peroxisome morphology in yeast mutants lacking either or both genes.
GFP-PTS1 was used to mark the peroxisomes. Also, we measured peroxisome
abundance in these strains during growth on glucose, which tends to
repress peroxisome abundance, and oleic acid, which induces peroxisome abundance (in yeast, peroxisomes are the sole sites of fatty acid
-oxidation and are required for growth on fatty acids such as oleic
acid (36)).
As noted previously, the abundance of peroxisomes in the S. cerevisiae strain BY4733 is extremely heterogeneous (17). This was
observed under a variety of growth conditions and complicated the
quantification of peroxisome abundance. However, even with this
variability, it is clear that the peroxisome abundance increased when
cells were shifted from glucose-containing medium to oleic acid-containing medium (Fig. 7,
A and B). As
reported many times in previous studies, loss of PEX11 (XLY1)
eliminated the increase in peroxisome abundance which occurs in
response to fatty acids (Fig. 7D). However, it caused only a
very slight reduction in basal peroxisome abundance during growth on
glucose (Fig. 7C). Loss of Vps1 (XLY3), in contrast,
severely reduced basal peroxisome abundance (Fig. 7E) and
also eliminated fatty acid-induced peroxisome proliferation (Fig.
7F), as shown by the earlier study by Hoepfner et
al. (19). Even though there were very few peroxisomes in vps1
mutants, peroxisome abundance appeared to be reduced
even further by the loss of both PEX11 and VPS1 (XLY4) (Fig. 7,
G and H). We also tested whether artificial
overexpression of PEX11 might be able to drive peroxisome division in
the absence of Vps1p. For this we transformed the vps1
strain with two different plasmids: an empty vector with a
GAL1 promoter or the same vector with the PEX11
open reading frame driven by the GAL1 promoter, which was able to
induce peroxisome proliferation in the XLY1 strain (Fig. 7I). After 24-h growth in galactose medium, we found that
peroxisome abundance was similar in both strains (Fig. 7, J
and K), providing further evidence that the PEX11-mediated
peroxisome proliferation requires Vsp1/DLP1.

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Fig. 7.
Yeast Vps1p is required for
PEX11-mediated peroxisome proliferation. Various S. cerevisiae strains were cultured under different conditions, and
the peroxisome number was counted under a fluorescence microscope in
120 cells for each sample. Shown here are the frequency distributions
of cells with different peroxisome numbers. A and
B, BY4733 cells transformed with
pPGK1-GFP/PTS1 and
pRS425/GAL1 were precultured in S minimal medium with 2%
glucose to midlog phase (A), shifted to minimal medium
containing 0.2% oleic acid and 0.02% Tween 40, and cultured for
24 h (B). C and D, XLY1
(pex11 ) cells carrying pRS425/GAL1 were
precultured in minimal S medium with 2% glucose to midlog phase
(C) and shifted to minimal medium containing 0.2% oleic
acid and 0.02% Tween 40 (D) for 24 h. E and
F, XLY3 (vps1 ) cells carrying
pRS425/GAL1 were precultured in minimal S medium with 2%
glucose to midlog phase (E) and shifted to minimal medium
containing 0.2% oleic acid and 0.02% Tween 40 (F) for
24 h. G and H, XLY4 (pex11 ,
vps1 ) cells carrying pRS425/GAL1 were precultured in
minimal S medium with 2% glucose to midlog phase (G) and
shifted to minimal medium 0.2% oleic acid and 0.02% Tween 40 (H) for 24 h. I, J, and
K, galactose-induced XLY1 cells transformed
pRS425/GAL1-PEX11 (I), XLY3 cells (J),
and XLY3 cells transformed with pRS425/GAL1-PEX11
(K). The desired yeast strains were cultured in glucose to
midlog phase, shifted to minimal medium containing 1% galactose, and
cultured for 24 h. Ola, oleic acid.
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No Evidence for Physical Interaction between DLP1 and
PEX11--
We have shown that DLP1 participates in PEX11-mediated
peroxisome proliferation, possibly by mediating peroxisome division. Next we set out to determine whether there is a physical association between DLP1 and PEX11 proteins. We first tested conditions for solubilizing PEX11 proteins from the peroxisome membrane. PEX11
and
PEX11
were efficiently solubilized by both 1% Triton X-100 and
0.2% digitonin (Fig. 8A). We
then tested whether PEX11 proteins solubilized under these conditions
retained the ability to interact with other proteins. Specifically, we
transfected cells with plasmid pairs expressing the following proteins:
PEX11
-3×HA and PEX11
myc (a), PEX11
-3×HA and
PEX11
(b), PEX11
-3×HA and PEX11
myc (c), PEX11
-3×HA and PEX11
(d), PEX11
-3×HA and
PEX11
myc (e), or PEX11
-3×HA and PEX11
(f). After a 2-day incubation, each cell population was
lysed with either 1% Triton X-100 or 0.2% digitonin, processed for
immunoprecipitation using anti-myc polyclonal antibodies, and the
immunoprecipitates were separated by SDS-PAGE and probed with anti-HA
antibodies (Fig. 8A). Although the PEX11 proteins were
solubilized by either detergent, we only observed PEX11
-PEX11
and
PEX11
-PEX11
interactions in cells homogenized with 0.2% digitonin. No protein-protein interactions were detected between PEX11
and PEX11
. These findings are in agreement with our
previous observation that Triton X-100 impairs the detection of PEX11
proteins by immunofluorescence microscopy (12), implying that PEX11
might be one of the rare proteins whose structure is sensitive to
Triton X-100.

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Fig. 8.
DLP1 does not physically interact with
PEX11. A, 5756-Ti cells expressing PEX11 -3×HA and
PEX11 myc (a), PEX11 -3×HA and PEX11 (b),
PEX11 -3×HA and PEX11 myc (c), PEX11 -3×HA and
PEX11 (d), PEX11 -3×HA and PEX11 myc (e),
or PEX11 -3×HA and PEX11 (f) were lysed in TBS buffer
with 1% Triton X-100 or 0.2% digitonin, and the lysates were cleared
by centrifugation at 100,000 × g for 30 min. The
resulting supernatant was processed for immunoprecipitation
(IP) using anti-myc polyclonal antibodies. The supernatant
and corresponding immunoprecipitation sample were then separated by
SDS-PAGE and probed with anti-HA monoclonal antibodies. Samples
b, d, and f are the negative controls
of samples a, c, and e, respectively.
B, 5756-Ti cells expressing PEX11 -3×HA and mycDLP1a
(a), PEX11 -3×HA and mycDLP1a (b),
PEX11 -3×HA and mycDLP1a (c), or PMP34-3×HA and mycDLP1a
(d) were lysed in TBS buffer with 0.2% digitonin, and the
lysates were cleared by centrifugation at 100,000 × g
for 30 min. The resulting supernatant was processed for
immunoprecipitation using anti-HA monoclonal antibodies. The
supernatant and corresponding immunoprecipitate sample were then
separated by SDS-PAGE and probed with anti-myc polyclonal
antibodies.
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Using the conditions that allow detection of homophilic interaction
between PEX11 proteins, we next tested whether DLP1 physically associated with PEX11. Fibroblasts were transfected with plasmids expressing PEX11
-3×HA and mycDLP1a (a), PEX11
-3×HA
and mycDLP1a (b), PEX11
-3×HA and mycDLP1a
(c), or PMP34-3×HA and mycDLP1a (d), incubated
for 24 h, lysed in 0.2% digitonin, and subjected to
immunoprecipitation using anti-HA antibodies. The immunoprecipitate samples were then fractionated by SDS-PAGE and blotted with anti-c-myc antibodies. No mycDLP1 was detected in any of these samples (Fig. 8B). Additional immunoprecipitation experiments using
cross-linkers and nonhydrolyzable GTP analogs also failed to show any
evidence of PEX11-DLP1 interactions (data not shown). Finally, we used a yeast two-hybrid system to test for possible interactions between these DLP1 and PEX11, but this too failed to show any evidence for
PEX11-DLP1 interaction (data not shown).
Splice Variants of DLP1 Differ in Their Effects on Peroxisome
Dynamics--
Multiple forms of DLP1 are generated by alternative
splicing of the initial DLP1 transcript, and it has been
proposed that some of these may have different activities in
vivo (20, 33). We tested whether any of the four splice variants
altered the activity of DLP1 vis à vis peroxisome
dynamics. These four forms of DLP1 differ as shown in Fig.
9A, with the top form (isoform a) being the one used in preceding experiments. When
expressed in human fibroblasts, isoform a had no significant
effect on peroxisome abundance (Fig. 9, B-D). Isoform
b also had no discernible effect on peroxisome abundance
(Fig. 9, B, E, and F). By contrast,
isoforms c and full-length DLP1 appeared to inhibit
peroxisome division (Fig. 9, B and G-J). The
S39N mutants of all four isoforms all inhibited peroxisome division
(Fig. 9B).

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Fig. 9.
Splice variants of DLP1
differ in their effects on peroxisome dynamics.
A, schematic summary of the alternative splicing region of
DLP1. Numbers indicate amino acid positions. Three different
isoforms, DLP1a, DLP1b, and DLP1c, are
cloned from fibroblasts. B, peroxisome abundance in 5756-Ti
cells, and 5756-Ti cells expressing four different isoforms of DLP1 and
their corresponding S39N mutants. C-J, representative cells
transfected with pcDNA3-mycDLP1a (C and
D), pcDNA3-mycDLP1b (E and
F), pcDNA3-mycDLP1c (G and
H), or pcDNA3-mycDLP1F (I and
J). 5756-Ti cells were transfected with appropriate
plasmids. 4 days after transfection, cells were processed for indirect
immunofluorescence with antibodies to the myc epitope (C,
E, G, and I) and PMP70 (D,
F, H, and J).
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DISCUSSION |
Until recently, the only factors implicated in peroxisome division
were those of the PEX11 family of peroxisomal membrane proteins. The
levels of PEX11 proteins correlate roughly with peroxisome abundance in
the cell, and overexpression of PEX11 alone is sufficient to accelerate
peroxisome division and greatly increase peroxisome abundance. However,
PEX11 does not share any homology to proteins involved in other
organelle formation processes, making it difficult to develop
hypotheses for PEX11 function. PEX11 overexpression induces peroxisome
division, but loss of PEX11 does not block peroxisome division entirely
and results in only a partial reduction in peroxisome abundance, in
both mammalian and yeast cells (7, 8, 14). Thus, it is likely that
PEX11 proteins play facilitative roles in peroxisome division rather than a core, conserved role in the biochemistry of peroxisome division.
In this study we extended the previous observation by Hoepfner
et al. (19) that Vps1p, a dynamin-like protein of yeast, also plays a critical role in peroxisome division. Like so many interesting observations, those of Hoepfner et al. (19)
raised many questions. Were similar proteins required for peroxisome division in other organisms? Do these proteins associate with peroxisomes? Does PEX11 recruit these proteins to peroxisomes? How do
dynamin-like proteins mediate peroxisome division? In this report we
have established that DLP1 plays an essential role in peroxisome
division in human cells and does associate with peroxisomes. Most
importantly, however, our data suggest a model of peroxisome division
in which elevated levels of PEX11 appear to induce peroxisome division
by recruiting DLP1 to peroxisomes.
We observed previously that there are two phases to PEX11-induced
peroxisome division in human cells (12, 17). The first phase involves
peroxisome elongation and the segregation of PEX11 from other
components of the peroxisome membrane. The second phase involves the
division of these elongated, tubular peroxisomes into numerous small
peroxisomes scattered throughout the cell. We detected small amounts of
DLP1 on peroxisomes under normal conditions, but overexpression of
PEX11 nearly doubled the level of peroxisome-associated DLP1.
Furthermore, immunofluorescence experiments revealed that many of the
elongated peroxisomes induced by PEX11 overexpression had small foci
that stained for DLP1. These results suggest that PEX11 induces
peroxisome division, at least in part, by recruiting DLP1 to peroxisome
membranes. Consistent with this hypothesis, we found that loss of DLP1
blocks PEX11-mediated peroxisome division but does not affect
PEX11-mediated elongation of peroxisomes or the formation of
PEX11-enriched patches at foci along the length of the elongated
peroxisomes. It should be noted, however, that PEX11 is unlikely to be
the sole mediator of DLP1 recruitment to membranes because loss of
PEX11 does not result in as severe a defect in peroxisome abundance as
loss of DLP1 in human cells or loss of VPS1 in yeast.
Although the data appear to favor a model of peroxisome division in
which PEX11 recruits DLP1 to peroxisome membranes, perhaps at sites of
PEX11 enrichment, we found no evidence for recruitment via direct
protein-protein interactions between PEX11 and DLP1. Such negative data
might reflect experimental artifacts that are not physiologically
relevant. However, these results at least raise the possibility that
PEX11 acts indirectly to facilitate DLP1 recruitment. Lipid moieties
have been implicated in dynamin recruitment to membranes (37-39), and
the relative levels of PEX11 might affect peroxisomal membrane
composition or the presence of lipid microdomains of distinct
composition which could act as sites of recruitment. Although DLP1
lacks the known lipid binding domain of dynamin, DLP1 might interact
with lipids in a fundamentally different but equally important manner.
An indirect mechanism for PEX11-mediated DLP1 recruitment is also
easier to reconcile with our observation of PEX11-independent,
DLP1-dependent peroxisome division by allowing other
factors to modulate the recruitment of DLP1 to peroxisomes. As for the
kinetics of the DLP1-peroxisome interaction, a few lines of evidence
suggest that it is transient. First, the steady-state proportion of
DLP1 which is associated with peroxisomes is very low and rises only
slightly (1.5-fold) by pronounced PEX11 overexpression. Second, DLP1 is
detected on only a small number of peroxisomes under normal conditions,
and after PEX11 overexpression it is detected preferentially on
elongated peroxisomes prior to division. Third, when we locked DLP1 on
peroxisomes by fusion to the cytoplasmic tail of an integral
peroxisomal membrane protein, peroxisome division was severely
inhibited (data not shown), indicating that DLP1 release is an
important step in peroxisome division. A transient interaction model
for DLP1 is similar to that proposed for dynamin in the formation of
clathrin-coated vesicles, where dynamin is recruited to the membrane
just prior to vesicle formation and is released immediately thereafter
(40).
As for how DLP1 actually catalyzes peroxisome division, our paper
offers little in the way of new insight into the function of
dynamin-like proteins. Dynamin has been proposed to act as a
pinchase, constricting the neck of nascent vesicles to form a
membranous tube, which presumably promotes fusion of the tightly apposed membrane bilayers (35, 41). However, dynamin does not appear to
be sufficient for completing the vesicle scission event, and other
factors, such as endophilin, might contribute essential membrane/lipid
modifying activities that mediate the final step in vesicle formation
(42-44). A similar mechanism might explain the role of DLP1 in
peroxisome division. Regardless of the actual mechanism, it is clear
that further advances in this field will require the development of
techniques that allow the measurement of peroxisome division by real
time video microscopy. Extensive efforts on our part to perform such
experiments have yet to yield success. The problems in detecting
peroxisome division are related to the relatively small size of
peroxisomes, their movement, and the relatively low rate at which they
divide. Based on our experience, we feel that successful monitoring of
peroxisome division might require the identification of inhibitory
drugs that can be used to synchronize peroxisomes in the predivision, elongated state, allowing investigators to follow a wave of peroxisome division once such inhibitors were removed.