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Mol Cell Biol, May 1998, p. 2789-2803, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutants of the Yeast Yarrowia lipolytica
Defective in Protein Exit from the Endoplasmic Reticulum Are Also
Defective in Peroxisome Biogenesis
Vladimir I.
Titorenko and
Richard A.
Rachubinski*
Department of Cell Biology and Anatomy,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Received 23 September 1997/Returned for modification 21 November
1997/Accepted 26 February 1998
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ABSTRACT |
Mutations in the SEC238 and SRP54 genes of
the yeast Yarrowia lipolytica not only cause
temperature-sensitive defects in the exit of the precursor form
of alkaline extracellular protease and of other secretory
proteins from the endoplasmic reticulum and in protein secretion
but also lead to temperature-sensitive growth in oleic acid-containing
medium, the metabolism of which requires the assembly of functionally
intact peroxisomes. The sec238A and
srp54KO mutations at the restrictive temperature
significantly reduce the size and number of peroxisomes,
affect the import of peroxisomal matrix and membrane proteins into the
organelle, and significantly delay, but do not prevent, the exit of
two peroxisomal membrane proteins, Pex2p and Pex16p, from the
endoplasmic reticulum en route to the peroxisomal membrane. Mutations
in the PEX1 and PEX6 genes, which encode
members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also
prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p
from the endoplasmic reticulum and cause the
accumulation of an extensive network of endoplasmic
reticulum membranes. None of the peroxisomal matrix proteins tested
associated with the endoplasmic reticulum in sec238A,
srp54KO, pex1-1, and pex6KO mutant
cells. Our data provide evidence that the endoplasmic reticulum is
required for peroxisome biogenesis and suggest that in
Y. lipolytica, the trafficking of some membrane
proteins, but not matrix proteins, to the peroxisome
occurs via the endoplasmic reticulum, results in their
glycosylation within the lumen of the endoplasmic
reticulum, does not involve transport through the Golgi, and requires
the products encoded by the SEC238, SRP54,
PEX1, and PEX6 genes.
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INTRODUCTION |
One of the hallmarks of
eukaryotic cells is the coexistence of functionally distinct
subcellular organelles (compartments), with each organelle possessing a
specific set of enzymes required for its particular metabolic role. One
organelle, the peroxisome, is present in most eukaryotic
cells (22). Peroxisomes compartmentalize more than 50 enzymes involved in different metabolic functions, including the
-oxidation of fatty acids and the decomposition of
H2O2 by catalase (42, 49). The
importance of peroxisomes for normal human development and
physiology is demonstrated by the lethality of various
peroxisome biogenesis disorders (23).
Changes in the abundance and composition of peroxisomes in
response to changes in environmental conditions must be coordinated with the biogenesis and functioning of other organelles in order to
achieve an overall balance in cellular function. An example of such
interorganellar communication is the tripartite path of communication
among mitochondria, the nucleus, and peroxisomes, which
regulates the expression of genes encoding peroxisomal proteins (6, 32). Some peroxisomal proteins may also play an
important role in the biogenesis of other organelles. The
peroxisome-associated protein Car1p has been shown to be
essential for karyogamy in the filamentous fungus Podospora
anserina (3). A functional relationship between
peroxisome biogenesis and a specific process in cell
morphogenesis, i.e., the dimorphic transition from the yeast to the
mycelial form, has also been demonstrated recently in the yeast
Yarrowia lipolytica (46).
While the role of the endoplasmic reticulum (ER) as the entry point for
all compartments of the secretory and endocytic pathways is well
established (27, 36, 41), the significance of the ER for
peroxisome biogenesis remains unclear. Recent data have suggested a dual role for the ER in peroxisome biogenesis
in supplying phospholipid for the formation of the peroxisomal membrane
(45) and in protein trafficking to peroxisomes
(2, 4, 13, 50, 51). We have applied a combined genetic,
biochemical, and morphological approach to study the importance of the
ER for peroxisome biogenesis in Y. lipolytica. An analysis of Y. lipolytica sec238A,
srp54KO, pex1-1, and pex6KO mutants
that are deficient in the exit of secretory proteins from the ER, in
protein secretion, and in peroxisome biogenesis has
provided evidence for an essential role for the ER in the assembly of
peroxisomes.
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MATERIALS AND METHODS |
Yeast strains and microbial techniques.
The Y. lipolytica strains used in this study are listed in Table
1. The new nomenclature for
peroxisome assembly genes and proteins has been used
(8). Media, growth conditions, and genetic techniques for
Y. lipolytica have been described (33, 35, 43). Medium components were as follows. YEPD contains 1% yeast extract, 2% peptone, and 2% glucose. 2×-YEPD contains 2% yeast extract, 4% peptone, and 4% glucose. YPBO contains 0.3% yeast extract, 0.5% peptone, 0.5% K2HPO4,
0.5% KH2PO4, 1% Brij 35, and 1%
(wt/vol) oleic acid. 2×-YPBO contains 0.6% yeast extract, 1% peptone, 1% K2HPO4, 1%
KH2PO4, 2% Brij 35, and 2% (wt/vol) oleic acid. YND contains 0.67% yeast nitrogen base without amino acids and
2% glucose. YNO contains 0.67% yeast nitrogen base without amino
acids, 0.05% (vol/vol) Tween 40, and 0.1% (wt/vol) oleic acid. YND
and YNO were supplemented with adenine, leucine, histidine, and lysine,
each at 50 µg/ml, as required.
Electron and immunofluorescence microscopy.
Electron
microscopy (16) and double-labeling, indirect
immunofluorescence microscopy (43) were performed as
previously described.
Subcellular fractionation.
The initial step in the
subcellular fractionation of YPBO-grown cells was performed as
described previously (43) and included the differential
centrifugation of lysed and homogenized spheroplasts at 1,000 × gmax for 8 min at 4°C in a Beckman JS13.1
rotor to yield a postnuclear supernatant (PNS) fraction. The PNS
fraction was further subjected to differential centrifugation at
20,000 × gmax for 30 min at 4°C in a
Beckman JS13.1 rotor to yield pellet (20KgP) and supernatant (20KgS)
fractions. The 20KgS fraction was further subfractionated by
differential centrifugation at 245,000 × gmax for 1 h at 4°C in a Beckman TLA120.2
rotor to yield pellet (245KgP) and supernatant (245KgS) fractions.
Protease protection analysis of different subcellular fractions was
performed as previously described (45). Separation of the
organelles, i.e., peroxisomes, plasma membrane,
mitochondria, ER, Golgi, and vacuoles, present in the 20KgP fraction of
YPBO-grown cells was performed by isopycnic centrifugation on a
discontinuous sucrose (25, 35, 42, and 53%, wt/wt) gradient in a
Beckman VTi50 rotor at 100,000 × gmax for 1 h at 4°C.
Purification of peroxisomes (
43,
45), ER
(
46), and plasma membranes (
46) and in vitro
disassembly of the complex formed
by peroxisomes and the ER
in mutant strains (
45) have already
been described.
Peroxisomes and the ER were more than 97% pure,
as determined by
marker protein analyses. Contamination of purified
peroxisomes by ER elements and vice versa was less than
0.5%.
Radiolabeling, immunoprecipitation, and endo H digestion.
Yeast cultures were grown in YPBO or YEPD at the temperatures indicated
in Results. Aliquots of cells were sedimented in a clinical centrifuge,
resuspended at a concentration of 2 U of optical density at 600 nm
(OD600)/ml in YNO (YPBO grown) or YND (YEPD grown) medium,
and incubated at the temperatures indicated for 30 min. Radiolabeling
was performed in the same medium containing L-[35S]methionine (ICN Biomedicals,
Mississauga, Ontario, Canada) at a concentration of 40 µCi/OD600 U for 3 min (in YNO) or 1.5 min (in YND) at the
temperatures indicated and chased with an equal volume of 2×-YPBO or
2×-YEPD, respectively, supplemented with 10 mM
L-methionine. Samples were taken at the postchase times indicated in Results. Reactions were terminated by the addition of an
equal volume of ice-cold 20 mM NaN3, and cells were
immediately separated from the culture supernatant by centrifugation in
a microcentrifuge at 20,000 × gmax for 3 min at 4°C. Immunoprecipitation of pulse-labeled Kar2p and Sec14p
from fractions of discontinuous sucrose gradients was performed as
previously described (52). Digestion of immunoprecipitated,
pulse-labeled Pex2p, Pex16p, thiolase (THI), acyl coenzyme A oxidase
(AOX), and isocitrate lyase (ICL) with endoglycosidase H (endo H) was
performed as previously described (26). Samples were
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Gels were treated with 22.2% 2,5-diphenyloxazole in either
dimethyl sulfoxide or glacial acetic acid (7), dried, and
exposed to preflashed Kodak X-Omat AR X-ray film at 
80°C with
intensifying screens.
Antibodies.
Guinea pig polyclonal antibodies to
Y. lipolytica ICL, THI, Pex2p, Pex5p, and Pex16p and to
Saccharomyces cerevisiae AOX and rabbit polyclonal anti-SKL
antibodies have already been described (11, 12, 43). Rabbit
polyclonal antibodies to S. cerevisiae malate synthase (MLS)
(12) and to Y. lipolytica alkaline
extracellular protease (AEP) (26), Sls1p (5),
Kar2p (46), and Sec14p (25) were described
previously. Anti-MLS antibodies were kindly provided by Andreas Hartig
(Institute of Biochemistry and Molecular Cell Biology, Vienna,
Austria). Anti-AEP and anti-Kar2p antibodies were generous gifts of
David Ogrydziak (University of California, Davis). Anti-Sls1p and
anti-Sec14p antibodies were generous gifts of Claude Gaillardin
(Institut National Agronomique Paris-Grignon, Thiveral-Grignon,
France). Mouse monoclonal antibody SPA-827 specific for grp78 (BiP) was
from StressGen Biotechnologies (Victoria, British Columbia, Canada).
Analytical procedures.
Enzymatic activities of catalase,
cytochrome c oxidase (43), NADPH:cytochrome
c reductase, 
-mannosidase, vanadate-sensitive plasma membrane ATPase (39), guanosine diphosphatase
(1), and alkaline phosphatase (44) were
determined by established methods. Inorganic phosphate liberated in
assays for the activities of guanosine diphosphatase and
vanadate-sensitive plasma membrane ATPase was measured as previously
described (21). SDS-PAGE (20) and immunoblotting
using a semidry electrophoretic transfer system (ET-20; Tyler Research
Instruments, Edmonton, Alberta, Canada) (19) were performed
as previously described. Antigen-antibody complexes were detected by
enhanced chemiluminescence (Amersham Life Sciences, Oakville, Ontario,
Canada). Quantitation of immunoblots was performed as previously
described (43).
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RESULTS |
The sec238A and srp54KO mutations cause
temperature-sensitive growth in oleic acid-containing medium.
A
characteristic feature of Y. lipolytica is its
extensive peroxisome proliferation during growth in oleic
acid-containing medium. The assembly of functionally intact
peroxisomes is absolutely required for growth in media
containing oleic acid as the sole carbon source (33). In
contrast, growth in glucose-containing medium does not require intact
peroxisomes (33). We have previously demonstrated that sec238A and srp54KO mutants
grown in glucose-containing YEPD medium show temperature-sensitive
defects in protein secretion (46). To study the possible
effects of the sec238A and srp54KO mutations on
peroxisome biogenesis, we first tested the ability of
sec238A and srp54KO mutants to grow in oleic
acid-containing YPBO medium at either 22 or 32°C (Fig.
1). Both strains were affected in growth
in YPBO only at 32°C, with doubling times of 11.0 and 10.3 h for
the sec238A and srp54KO mutants, respectively,
compared to 2.5 h for the isogenic wild-type strain. No effect of
either the sec238A or the srp54KO mutation on
growth in YPBO at 22°C was observed. Neither the sec238A
nor the srp54KO mutation affected growth in YEPD medium at
either 22 or 32°C.
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FIG. 1.
The sec238A and srp54KO mutations
cause temperature-sensitive growth in YPBO but do not affect growth in
YEPD. Growth curves and doubling times (td) of wild-type
(WT) strain DX547-1A and of the sec238A and
srp54KO mutants grown in YEPD or YPBO at 22°C (×) or
32°C ( ). Cells were pregrown twice in YEPD at 22 or 32°C until
an OD600 of 1.8 to 2.0 (cell titer, ~1.1 × 108 to 1.3 × 108 cells/ml) and inoculated
into YEPD or YPBO at an initial OD600 of 0.1 (cell titer,
~6.1 × 106 to 6.5 × 106
cells/ml).
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The sec238A and srp54KO mutations
significantly reduce the size and number of peroxisomes at
the restrictive temperature.
We have previously reported
(46) that the sec238A and
srp54KO mutations affect protein secretion and exit of the
precursor form (pAEP) of AEP, a major secretory protein of
Y. lipolytica (14, 17, 26), from the ER in
YEPD medium at the restrictive temperature of 32°C (46).
Similar results were observed for the sec238A and
srp54KO mutations on a carbon source, oleic acid, which can
be metabolized only by functionally intact peroxisomes (data not shown). These results, combined with the observation that the
sec238A and srp54KO mutant strains are retarded
in their growth in YPBO medium at 32°C (Fig. 1), suggested to us that
these two temperature-sensitive mutations not only compromise the exit of secretory proteins from the ER and their delivery to the
extracellular medium but also might affect some aspect(s) of
peroxisome biogenesis.
Electron microscopical analysis showed no significant differences in
peroxisome size and number between the wild-type and
mutant
strains grown in YPBO at 22°C, the temperature permissive
for growth
and protein secretion (Fig.
2A). However,
the size
and number of peroxisomes in
sec238A
and
srp54KO mutant cells
at the restrictive temperature,
32°C, were significantly reduced
compared to those of wild-type cell
peroxisomes (Fig.
2A). The
sec238A and
srp54KO mutant cells also accumulated 100- to 120-nm
vesicles (Fig.
2A, arrowheads) at the restrictive temperature.
These
vesicles were rarely observed in wild-type cells (Fig.
2A).
Moreover,
in contrast to the peroxisomes in wild-type cells,
peroxisomes
in
sec238A and
srp54KO
mutant cells at either 22 or 32°C were
closely apposed to ER
membranes (Fig.
2A).
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FIG. 2.
The sec238A and srp54KO mutations
significantly reduce the size and number of peroxisomes at
the restrictive temperature. (A) Cells were grown for 9 h in YPBO
at either 22 or 32°C, fixed in KMnO4, and processed for
electron microscopy. Abbreviations: P, peroxisome; M,
mitochondrion; N, nucleus; WT, wild type. Arrowheads, 100- to 120-nm
vesicles accumulating in sec238A and srp54KO
mutant cells at 32°C. Bars, 1 µm. (B to D) Results of morphometric
analyses performed on random electron microscopic sections of cells of
wild-type and mutant strains grown in YPBO at either 22 or 32°C. (B
and C) Percentages of peroxisomes having the indicated
relative areas of peroxisome section. The relative area of
peroxisome section is calculated as the area of
peroxisome section/area of cell section × 100. (D)
Numbers of peroxisomes in wild-type and mutant strains.
Peroxisomes were counted in electron micrographs, and the data were
expressed as the number of peroxisomes per cubic micrometer
of cell section volume. The bar in the middle of each box indicates the
mean peroxisome number. The limits of each box indicate the
sample standard deviation on each side of the mean. The lines extend to
indicate the extremes of the distribution.
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Morphometric analysis of random cell sections of the wild-type and
mutant strains grown in YPBO at either 22 or 32°C showed
that the
sec238A and
srp54KO mutations significantly
reduced the
size of peroxisomes at the restrictive
temperature compared to
the wild-type condition. In
sec238A
and
srp54KO cells, there was
an accumulation of exclusively
(
sec238A) or mostly (
srp54KO) small
peroxisomes at the restrictive temperature of 32°C, with
their
relative areas of peroxisome section ranging from 0.3 to 1.0%
(Fig.
2C). In wild-type cells at 32°C, more than half
(54.7%)
of all peroxisomes had relative areas of
peroxisome section ranging
from 1.1 to 2.0%, while 25.7%
of all peroxisomes had relative
areas of
peroxisome section ranging from 2.1 to 5.0% (Fig.
2C).
The
effect of either mutation on peroxisome size was much less
pronounced at the permissive temperature (Fig.
2B).
The
sec238A and
srp54KO mutations also showed a
four- to fivefold decrease in the number of peroxisomes per
cell at the restrictive
temperature (3.18 ± 2.45 and 4.9 ± 0.83 peroxisomes per µm
3 of cell section
volume, respectively) compared to the number
of peroxisomes
in wild-type cells (19.84 ± 3.53 peroxisomes per
µm
3 of cell section volume) (Fig.
2D). The effect of
either mutation
on the number of peroxisomes per cell was
much less pronounced
at the permissive temperature (14.88 ± 4.34, 11.53 ± 5.24, and
11.56 ± 3.36 peroxisomes per
µm
3 of cell section volume for the wild-type,
sec238A, and
srp54KO strains, respectively) (Fig.
2D).
The sec238A and srp54KO mutations affect
the subcellular localization of peroxisomal matrix and membrane
proteins at the restrictive temperature.
A significant
decrease in the size and number of peroxisomes, which was
observed in the sec238A and srp54KO mutants
grown in YPBO at the restrictive temperature of 32°C, suggested that these mutations either compromise the synthesis of peroxisomal proteins
or affect their import into the peroxisome at the
restrictive temperature, thereby leading to the mislocalization of
peroxisomal matrix and membrane proteins. When the wild-type,
sec238A, and srp54KO strains were shifted from
YEPD to YPBO and incubated for 8 h at 32°C, the levels of all
peroxisomal proteins were greatly induced and reached a steady state in
all three strains. No appreciable differences in the rate of induction
and in the steady-state levels of all peroxisomal matrix (AOX, MLS, 62- and 64-kDa anti-SKL-reactive polypeptides, ICL, THI, catalase, and
multifunctional enzyme) and membrane (Pex2p and Pex16p) proteins were
observed between the wild-type strain and both mutants at 32°C (data
not shown). Therefore, neither the sec238A nor the
srp54KO mutation affected the synthesis of peroxisomal
proteins at the temperature restrictive for the exit of secretory
proteins from the ER, protein secretion, and growth in oleic
acid-containing medium. In contrast, the distribution of peroxisomal
matrix and membrane proteins to different subcellular fractions was
altered in both mutants compared to the wild-type strain at the
restrictive temperature. In wild-type cells, the major fraction
(82 to 100%) of peroxisomal matrix and membrane proteins
was associated with the low-speed (20KgP) and high-speed (245KgP)
pelletable fractions (Fig. 3A and C show
the data for AOX, THI, and Pex2p; data for other proteins not shown).
These peroxisomal proteins were resistant to exogenously added protease (trypsin) and were therefore in membrane-enclosed structures (data not
shown). In sec238A and srp54KO mutant cells at
32°C, a large fraction (57 to 65%) of peroxisomal matrix proteins
was mislocalized to the 245KgS (cytosolic) fraction (Fig. 3A and C show
the data for AOX and THI; data for other peroxisomal matrix proteins
not shown). These proteins were accessible to external protease (data not shown). Some fraction of peroxisomal membrane proteins Pex2p and
Pex16p was also mislocalized to the cytosol in sec238A and srp54KO mutant cells at 32°C (Fig. 3C shows the data for
Pex2p; data for Pex16p not shown). However, the mislocalization of
Pex2p and Pex16p to the cytosol in both mutants was much less
pronounced than that of peroxisomal matrix proteins. Up to 15% of
Pex2p and up to 9% of Pex16p were mislocalized to the 245KgS fraction
in mutant cells (compare data for Pex2p to those for AOX and THI in
Fig. 3A and C; data for Pex16p not shown).
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FIG. 3.
The sec238A and srp54KO mutations
affect the subcellular localization of peroxisomal matrix and membrane
proteins at the restrictive temperature. The recoveries of the
peroxisomal matrix proteins AOX and THI and of the peroxisomal integral
membrane protein Pex2p in different subcellular fractions of the
wild-type (WT) and sec238A and srp54KO mutant
strains are presented. Strains were grown for 9 h in YPBO at
either 22 or 32°C and subjected to subcellular fractionation. (A)
Equal fractions (0.15% of the total volume for AOX and THI, 0.9% of
the total volume for Pex2p) of the PNS (lanes 1), 20KgP (lanes 2),
20KgS (lanes 3), 245KgP (lanes 4), and 245KgS (lanes 5) were analyzed
by immunoblotting with anti-AOX, anti-THI, and anti-Pex2p antibodies.
Immunoblots were scanned densitometrically, and the recovery of
peroxisomal proteins in different subcellular fractions was
quantitated. Values for the PNS (B), 20KgP plus 245KgP and 245KgS (C),
and 20KgP and 245KgP (D) signals are presented for the wild-type and
mutant strains grown in YPBO at 32°C. Values for signals in different
subcellular fractions of mutant strains in panels B, C, and D are
relative to the signal for a particular protein in corresponding
fractions of the wild-type strain grown at the same temperature. All
values for signals are means ± standard deviations for three
independent experiments.
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The relative distributions of pelletable, protease-protected pools of
THI (matrix protein) and of Pex2p and Pex16p (membrane
proteins)
between the 20KgP and 245KgP fractions were also altered
by the
sec238A and
srp54KO mutations at the restrictive
temperature.
In wild-type cells, the major fraction (70.6 to 73.8%) of
these
proteins was found in the 20KgP fraction, while 10.7 to 12.8%
was associated with the 245KgP fraction (Fig.
3D shows the data
for THI
and Pex2p; data for Pex16p not shown). In contrast, in
sec238A and
srp54KO mutant cells, pelletable
pools of THI, Pex2p,
and Pex16p were equally distributed between the
20KgP and 245KgP
fractions (Fig.
3D shows the data for THI and Pex2p;
data for
Pex16p not shown). This effect of the
sec238A and
srp54KO mutations
on the distribution of the pelletable
pools of peroxisomal proteins
between the high- and low-speed fractions
was due to the accumulation
of a population of high-speed-pelletable
vesicles containing THI,
Pex2p, and Pex16p but not AOX (Fig.
3D) or any
of the other peroxisomal
matrix proteins tested (data not shown). These
high-speed-pelletable
vesicles derive by budding from the ER and
initially contain Pex2p
and Pex16p. THI is imported into the vesicles
soon after they
bud from the ER and before they fuse with
peroxisomes (
48).
Data from immunofluorescence analysis were in agreement with the
results of subcellular fractionation and indicated that the
sec238A and
srp54KO mutations cause the
mislocalization of a large
fraction of peroxisomal matrix proteins
(THI, anti-SKL-reactive
proteins, ICL, and MLS) to the cytosol in cells
grown for 9 h
in YPBO at the restrictive temperature only (data
not shown).
The pex1-1 and pex6KO mutations cause the
accumulation of an extensive network of ER membranes and significantly
reduce the size and number of peroxisomes.
We have
previously shown that some, but not all, pex mutations,
i.e., the pex1-1, pex2KO, pex6KO, and
pex9KO mutations, significantly reduce the rate and
efficiency of protein secretion and affect the exit of secretory
proteins from the ER during growth in YEPD at both 22 and 32°C
(46). Similar results were obtained for these mutations
during growth in YPBO at both temperatures (data not shown). We
assessed the morphological effects of mutations in the PEX1
and PEX6 genes, which encode members of the AAA family of
N-ethylmaleimide-sensitive fusion protein-like ATPases
(8), during growth in YPBO. Electron microscopical analysis
showed an extensive proliferation of ER membranes in cells of
YPBO-grown pex1-1 and pex6KO mutants (Fig.
4A). The extent of ER membrane accumulation was assessed by comparing the lengths of ER membranes to
the cell circumference (relative length of ER membranes) in random
cross sections. While most (86.8%) of the wild-type cells had relative
ER membrane lengths ranging from 0.5 to 1.0, all pex1-1
mutant cells and most (90.9%) pex6KO mutant cells had
relative ER membrane lengths ranging from 1.5 to 3.0 (Fig. 4B).
Morphometric analysis of random sections of cells of the wild-type and
mutant strains also demonstrated that the pex1-1 and
pex6KO mutations significantly reduced the size of
peroxisomes. pex1-1 and pex6KO mutant
cells accumulated mostly small peroxisomes, with relative areas of peroxisome section ranging from 0.05 to 0.2%
(Fig. 4C). In contrast, in wild-type cells, more than one-third
(36.6%) of all peroxisomes had relative areas of
peroxisome section ranging from 1.0 to 1.5%, while 24.7%
of all peroxisomes had relative areas of
peroxisome section ranging from 1.5 to 3.0% (Fig. 4C). The
pex1-1 and pex6KO mutations also caused a three-
to fourfold decrease in the number of peroxisomes per cell
(10.78 ± 2.23 and 11.27 ± 2.71 peroxisomes per
µm3 of cell section volume, respectively) compared to the
number of peroxisomes in wild-type cells (41.65 ± 5.2 peroxisomes per µm3 of cell section volume)
(Fig. 4D).
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FIG. 4.
The pex1-1 and pex6KO mutations
cause the accumulation of ER membranes and significantly reduce the
size and number of peroxisomes. (A) Cells were grown for
9 h in YPBO at 32°C, fixed in KMnO4, and processed
for electron microscopy. Abbreviations are as defined in the legend to
Fig. 2. Bars, 1 µm. (B to D) Morphometric analysis was performed on
random electron microscopic sections of cells of the wild-type (WT) and
mutant strains grown in YPBO at 32°C. (B) The percentage of cells
having the indicated relative lengths of ER membranes. The relative
length of the ER membranes is calculated as the length of ER membranes
relative to cell circumference. (C) Percentage of
peroxisomes having the indicated relative area of
peroxisome section. The relative area of
peroxisome section is calculated as the [(area of
peroxisome section/area of cell section) × 100]. (D)
Numbers of peroxisomes in the wild-type and mutant strains.
Peroxisomes were counted in electron micrographs, and the data were
expressed as the number of peroxisomes per cubic micrometer
of cell section volume. The bar in the middle of each box indicates the
mean peroxisome number. The limits of each box indicate the
sample standard deviation on each side of the mean. The lines extend to
indicate the extremes of the distribution.
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The sec238A, srp54KO, pex1-1,
and pex6KO mutations cause accumulation of peroxisomal
membrane proteins Pex2p and Pex16p in the ER.
In wild-type
Y. lipolytica cells grown in oleic acid-containing
medium, Pex2p and Pex16p are, correspondingly, integral (12) and peripheral (11) peroxisomal membrane proteins.
Wild-type, sec238A, and srp54KO cells were grown
in YEPD at 22°C, shifted to YPBO, and incubated for 1 or 4 h at
32°C. Indirect immunofluorescence microscopy of YEPD- and YPBO-grown
wild-type cells with anti-Pex2p (Fig. 5A)
and anti-Pex16p (data not shown) antibodies showed a punctate pattern
of staining characteristic of peroxisomes. Double-labeling, indirect immunofluorescence with antibodies to Pex2p and to the ER
luminal protein Kar2p (Fig. 5A), or to Pex16p and to Kar2p (data not
shown), showed that in YEPD- and YPBO-grown wild-type cells, punctate
structures decorated by antibodies to peroxisomal proteins failed to
colocalize with structures recognized by antibodies to Kar2p.
Antibodies to Kar2p showed fluorescence patterns characteristic of the
ER, including bright staining of the perinuclear region and cell
periphery, and occasionally of filamentous extensions into the cytosol
(37, 40, 46). The fluorescence patterns generated by
antibodies to Pex2p and to Kar2p (Fig. 5A), or to Pex16p and to Kar2p
(data not shown), in sec238A and srp54KO mutant cells in YEPD at 22°C were similar to those seen in wild-type cells
and showed no colocalization of Pex2p and Pex16p with ER elements.
However, a shift of sec238A and srp54KO mutant
cells from YEPD to YPBO and incubation for 1 h at 32°C, the
temperature restrictive for the exit of pAEP from the ER and for
protein secretion (data not shown) (46), caused the
localization of a significant fraction of both Pex2p (Fig. 5A) and
Pex16p (data not shown) to the ER. The association of both peroxisomal
membrane proteins with the ER was transient in sec238A and
srp54KO mutant cells shifted to YPBO at 32°C. At 4 h
after the shift, antibodies to both Pex2p (Fig. 5A) and Pex16p (data
not shown) yielded only a punctate pattern of staining characteristic
of peroxisomes and did not decorate ER elements.
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FIG. 5.
Peroxisomal membrane proteins Pex2p and Pex16p
transiently or permanently accumulate in the ER of sec238A,
srp54KO, pex1-1, and pex6KO mutant
cells. (A) Wild-type (WT) strain DX547-1A and sec238A and
srp54KO mutant strains were grown in YEPD at 22°C until
the cell titer was 2.0 × 108 to 2.3 × 108 cells/ml. Cell cultures were separated into three
aliquots. Two aliquots were transferred to YPBO and incubated for 1 or
4 h at 32°C. The third aliquot was processed immediately for
double-labeling, indirect immunofluorescence using rabbit
anti-Y. lipolytica Kar2p and guinea pig
anti-Y. lipolytica Pex2p primary antibodies. Primary
antibodies were detected with fluorescein-conjugated goat anti-rabbit
immunoglobulin G and rhodamine-conjugated donkey anti-guinea pig
immunoglobulin G secondary antibodies. (B) Wild-type strain DX547-1A
and sec238A and srp54KO mutant strains were grown
in YEPD at 22°C until the cell titer was 2.0 × 108
to 2.3 × 108 cells/ml. Cells were transferred to YPBO
and incubated at 32°C. Aliquots of cells were taken at the times
indicated. The levels of Pex2p in whole-cell lysates of the wild-type
and mutant strains were determined. The lysate from 6 × 108 cells was applied to each lane. Blots were probed with
anti-Pex2p antibodies. Immunoblots were scanned densitometrically, and
the level of Pex2p in wild-type and mutant cells at the times indicated
was quantitated. (C) Wild-type strain DX547-1A and sec238A
and srp54KO mutant strains were grown in YEPD at 22°C
until the cell titer was 2.0 × 108 to 2.3 × 108 cells/ml. Cultures were separated into three aliquots.
Cells from one aliquot were immediately labeled with
L-[35S]methionine. The other two aliquots
were transferred to YPBO, incubated for 1 or 4 h at 32°C, and
then labeled with L-[35S]methionine.
Aliquots of cells from each of the three subcultures of the wild-type
and mutant strains were taken at the times indicated, and radiolabeling
was terminated by the addition of an equal volume of ice-cold 20 mM
NaN3 and 20 mM L-methionine. Pex2p was
immunoprecipitated from whole-cell lysates derived from 6 × 108 cells. Immunoprecipitates were resolved by SDS-PAGE and
visualized by fluorography. Fluorograms were quantitated by
densitometry. Values for the level of Pex2p in wild-type and mutant
cells at the indicated times of labeling are relative to the maximum
level of Pex2p in cells of the corresponding strain preincubated in
YPBO for 1 h at 32°C, which was set at 100%. The half-times
(t1/2) of synthesis of Pex2p by wild-type and
mutant cells were calculated. 0, 1, and 4 represent aliquots of a
particular strain either pregrown in YEPD at 22°C (0) or preincubated
in YPBO for 1 h (1) or 4 h (4) at 32°C before
radiolabeling. (D) Wild-type strain E122 and pex1-1 and
pex6KO mutant strains were grown in YEPD at 32°C until the
cell titer was 1.8 × 107 to 2.1 × 107 cells/ml. Cells were transferred to YPBO and incubated
for 9 h at 32°C. Wild-type and mutant cells were processed for
double-labeling, indirect immunofluorescence using rabbit
anti-Y. lipolytica Kar2p and guinea pig
anti-Y. lipolytica Pex2p or rabbit
anti-Y. lipolytica Kar2p and guinea pig
anti-Y. lipolytica Pex16p primary antibodies. Primary
antibodies were detected with fluorescein-conjugated goat anti-rabbit
immunoglobulin G and rhodamine-conjugated donkey anti-guinea pig
immunoglobulin G secondary antibodies.
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How can the transient association of Pex2p and Pex16p with the ER seen
by immunofluorescence of
sec238A and
srp54KO
mutant
cells shifted to YPBO at 32°C be explained? An analysis of the
kinetics of induction of Pex2p in cells incubated in YPBO indicated
that by 1 h after the shift from YEPD to YPBO, the steady-state
levels of Pex2p were increased 40-, 25-, and 31-fold in wild-type,
sec238A, and
srp54KO cells, respectively (Fig.
5B). In contrast,
by 4 h after the shift, no significant increase
in the steady-state
levels of Pex2p was observed in wild-type and
mutant cells compared
to the levels of Pex2p in cells induced for
1 h (Fig.
5B). Therefore,
the fluorescence patterns generated
by antibodies to Pex2p in
cells shifted from YEPD to YPBO and incubated
at 32°C for either
1 or 4 h reflected the intracellular
localization of the bulk
of Pex2p (69.1 to 97.6% of the total Pex2p,
as judged from the
data in Fig.
5B), which was synthesized 1 h
after the shift. Furthermore,
the rates of synthesis of Pex2p after the
shift from YEPD to YPBO
for 1 h dramatically increased and reached
half-times of 2.1,
3.0, and 2.7 min for the wild-type,
sec238A, and
srp54KO strains,
respectively
(Fig.
5C). In wild-type cells incubated in YPBO for
1 h, the rate
of synthesis of Pex2p was comparable to its rate
of exit from the ER,
which had a half-time of 3.0 min (see Fig.
6A and F). In contrast, in
sec238A and
srp54KO mutant cells incubated
in
YPBO for 1 h, the rates of synthesis of Pex2p were 7.1 to 8.8
times faster than its rates of exit from the ER (half-times of
21.2 and
23.7 min for the
sec238A and
srp54KO mutant
strains,
respectively; see Fig.
7A and G for data on the
sec238A mutant
strain). Therefore, the bulk of Pex2p in
sec238A and
srp54KO mutant
cells shifted to YPBO
for 1 h was associated with the ER, as detected
by
immunofluorescence (Fig.
5A). The Pex2p already present in
peroxisomes of
sec238A and
srp54KO mutant cells pregrown in YEPD
at 22°C represents
only a minor fraction (3.1 to 3.9%, as judged
from the data in Fig.
5B) of the total Pex2p in cells of both
mutants incubated in YPBO for
1 h at 32°C. These low levels of
Pex2p might not be sufficient
to generate the immunofluorescent
punctate pattern of staining
characteristic of peroxisomes, as
the bulk of Pex2p is
localized to the ER in
sec238A and
srp54KO mutant
cells incubated in YPBO for 1 h at 32°C. In contrast, in
sec238A and
srp54KO mutant cells incubated in
YPBO for 4 h, the
rates of synthesis of Pex2p reached half-times
of 27.9 and 21.1
min, respectively (Fig.
5C), and these rates were
comparable to
the rates of exit of Pex2p from the ER, which had
half-times of
21.2 and 23.7 min for the
sec238A (see Fig.
7A
and G) and
srp54KO (data not shown) mutant strains,
respectively. Under these conditions,
the bulk of Pex2p can exit from
the ER of
sec238A and
srp54KO mutant cells, and
therefore, Pex2p was localized by immunofluorescence
in punctate
structures characteristic of peroxisomes rather than
colocalized in elements containing the ER lumenal protein Kar2p
(Fig.
5A).
While the localization of both Pex2p and Pex16p to ER elements in the
sec238A and
srp54KO mutants at the restrictive
temperature
was transient, both proteins were permanently associated
with
the ER in
pex1-1 and
pex6KO mutant cells.
The fluorescence patterns
generated by antibodies to Pex2p and to
Kar2p, or to Pex16p and
to Kar2p, in
pex1-1 and
pex6KO mutant cells either grown in YEPD
at 22°C (data not
shown) or shifted to YPBO and incubated at 32°C
for 9 h (Fig.
5D) were superimposable and showed staining characteristic
of the ER
(cf. Fig.
5A).
Our data are suggestive of trafficking of Pex2p and Pex16p to the
peroxisomal membrane via the ER in wild-type cells. The
exit of both
peroxisomal membrane proteins from the ER at 32°C
is significantly
delayed, but not prevented, by the
sec238A and
srp54KO mutations and is completely blocked by the
pex1-1 and
pex6KO mutations at 22 and 32°C.
In contrast, the trafficking
of peroxisomal matrix proteins, including
THI, MLS, ICL, and anti-SKL-reactive
proteins, apparently does not
occur via the ER. Double-labeling,
indirect immunofluorescence with
antibodies to any of these peroxisomal
proteins and to Kar2p showed no
association of matrix proteins
with the ER in any mutant cells under
the conditions in which
a significant fraction of Pex2p and Pex16p
localized to the ER
(data not shown).
Membrane proteins Pex2p and Pex16p, but not matrix proteins,
traffic to peroxisomes via the ER.
We studied the
trafficking of peroxisomal membrane and matrix proteins by
immunoprecipitation of pulse-labeled and chased proteins from fractions
of discontinuous sucrose density gradients of 20KgP subcellular
fractions. In wild-type cells, the majority of pulse-labeled, unchased
Pex2p cofractionated with the ER marker (Kar2p; Fig.
6A and D, left) but not with markers of
the Golgi (Sec14p; Fig. 6A and E, left; data for guanosine
diphosphatase not shown), plasma membrane, mitochondria, or vacuoles
(data not shown). By 5 min of chase, most of this ER-associated Pex2p
was chased into fractions containing peroxisomal proteins (Fig. 6A to
C, left parts). The transit of Pex2p from the ER to the
peroxisome was complete by 10 min of chase (Fig. 6A, left).
In contrast, the majority of pulse-labeled Pex2p in pex1-1
and pex6KO mutant cells localized to ER elements even by 60 min of chase (Fig. 6A and D, second and third parts). Only a minor
fraction of pulse-labeled Pex2p in pex1-1 and
pex6KO mutant cells was chased into fractions containing
peroxisomal proteins (Fig. 6A to C, second and third parts). No
pulse-labeled Pex2p in either wild-type or mutant cells cofractionated
with markers of the Golgi (Sec14p; Fig. 6A and E; data for guanosine
diphosphatase not shown), plasma membrane, mitochondria, or vacuoles
(data not shown). Similar results were obtained for the
trafficking of Pex16p (data not shown). Immunoprecipitation of
pulse-labeled Pex2p and Pex16p from the 20KgP, 245KgP, and 245KgS
(cytosolic) fractions showed no cytosolic pools of pulse-labeled Pex2p
or Pex16p prior to the appearance of either protein in
peroxisomes, i.e., at the start of the chase in wild-type,
pex1-1, or pex6KO cells (Fig. 6A, right, shows
the data for Pex2p; data for Pex16p not shown). Therefore, Pex2p and
Pex16p found in peroxisomes derive from the ER rather than
being imported directly from the cytosol. Accordingly, the targeting of
both peroxisomal membrane proteins to the ER in wild-type,
pex1-1, and pex6KO cells is a prerequisite for
their import into peroxisomes and is not a targeting
pathway alternative to a cytosol-to-peroxisome targeting
pathway for Pex2p and Pex16p. These data extend the results of
double-labeling, indirect immunofluorescence (Fig. 5) and demonstrate
that the delivery of the membrane proteins Pex2p and Pex16p to the
peroxisome occurs via the ER, does not involve their
transport through the Golgi (or any other organelle) as an intermediate
step, and is largely prevented by the pex1-1 and
pex6KO mutations. In contrast, the trafficking of the
peroxisomal matrix proteins THI (Fig. 6B), AOX (Fig. 6C), and ICL (data
not shown) does not occur via the ER (or any other organelle). None of
these proteins was associated with the ER at any time in
either wild-type or mutant cells.
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FIG. 6.
The pex1-1 and pex6KO mutations
prevent the exit of Pex2p from the ER and differentially affect the
peroxisomal import of Pex2p, THI, and AOX. Wild-type (WT) and
mutant strains grown in YPBO at 32°C were pulse-labeled for 3 min
with L-[35S]methionine and chased with
unlabeled L-methionine. Samples were taken at the indicated
times postchase. Cells were subjected to subcellular fractionation, and
the 20KgP fractions were further fractionated by isopycnic
centrifugation on discontinuous sucrose density gradients. Cells taken
at chase time 0 were also subjected to subcellular fractionation to
yield 20KgP, 245KgP, and 245KgS (cytosolic) fractions. Pex2p (A), THI
(B), AOX (C), Kar2p (D), and Sec14p (E) were immunoprecipitated from
gradient fractions. Pex2p (A) was also immunoprecipitated from 20KgP,
245KgP, and 245KgS fractions of wild-type and mutant cells taken at
chase time 0. Immunoprecipitates were resolved by SDS-PAGE and
visualized by fluorography. Peak fractions in which protein
markers of peroxisomes (P), the ER, and the Golgi (G)
localized are indicated. (F) The first three panels of the fluorograms
in panel A were quantitated by densitometry. The levels of
ER-associated Pex2p in wild-type and mutant cells at the indicated
times postchase are relative to the level of ER-associated Pex2p in
wild-type cells at chase time 0, which was set at 100%. The half-times
(t1/2) for the exit of Pex2p from the ER of
wild-type and mutant cells were calculated. (G) The first three
parts of the fluorograms in panel A and the fluorograms in panels B and
C were quantitated by densitometry. The levels of Pex2p, THI, and AOX
imported into peroxisomes in wild-type and mutant cells at
the indicated chase times are relative to the levels of the respective
proteins imported into peroxisomes of wild-type cells after
a 60-min chase, which were set at 100%. The half-times for the import
of Pex2p, THI, and AOX into peroxisomes of wild-type and
mutant cells were calculated.
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In wild-type cells, Pex2p and Pex16p exited the ER with half-times
of 3.0 and 3.4 min, respectively, which were very close
to the
half-times for their import into peroxisomes (2.4 and 3.2
min, respectively) (Fig.
6F and G show the data for Pex2p; data
for
Pex16p not shown). These data suggest some coordination between
the
exit of Pex2p and Pex16p from the ER and their import into
the
peroxisome. The kinetics of import of Pex2p and Pex16p and
of the matrix protein THI into peroxisomes in wild-type
cells
were different from those of other peroxisomal proteins. While
the import of Pex2p (Fig.
6G), Pex16p (data not shown), and THI
(Fig.
6G) into peroxisomes in wild-type cells reached a steady
state by 10 min of chase, steady-state levels of peroxisomal import
for
AOX (Fig.
6G) and ICL (data not shown) were observed only
after 40 to
60 min of chase. Moreover, while both the
pex1-1 and
pex6KO mutations severely affected the import of Pex2p (Fig.
6G),
Pex16p (data not shown), and THI (Fig.
6G) into the
peroxisome,
their effects on the peroxisomal import of AOX
(Fig.
6G) and ICL
(data not shown) were much less pronounced. These
data suggest
that the import of Pex2p, Pex16p, and THI into the
peroxisome
could occur by a mechanism distinct from that
used for the import
of AOX and ICL. Indeed, the import of Pex2p,
Pex16p, and THI into
peroxisomes involves the budding from
the ER of vesicles that
initially contain Pex2p and Pex16p and import
THI after they bud
from the ER and before they fuse with
peroxisomes (
48).
We also studied the trafficking of peroxisomal membrane and matrix
proteins in
sec238A mutant cells grown in YPBO at 22 or
32°C. At 22°C, the temperature permissive for protein secretion,
the major portion of pulse-labeled, unchased Pex2p and Pex16p
cofractionated with the ER marker Kar2p (Fig.
7E and F,
left,
show the data for Pex2p and Kar2p; data for Pex16p not shown).
By
10 min of chase, all of Pex2p and Pex16p was chased into
fractions
containing peroxisomal proteins (Fig.
7E). Similar to the
data
presented above for wild-type,
pex1-1, and
pex6KO cells, no cytosolic
pool of either peroxisomal
membrane protein was found in
sec238A mutant cells at the
start of the chase, i.e., prior to the appearance
of Pex2p and Pex16p
in peroxisomes (Fig.
7E, right, shows the
data for Pex2p;
data for Pex16p not shown). Therefore, pulse-labeled
Pex2p and Pex16p
chased into the peroxisomal fractions of
sec238A mutant
cells grown at 22°C are not imported into peroxisomes
directly
from the cytosol but rather are delivered to
peroxisomes from
the ER. The trafficking of Pex2p and
Pex16p from the ER to the
peroxisome in
sec238A
mutant cells did not involve their transport
through the Golgi or any
other organelle, as no Pex2p (Fig.
7E,
left) or Pex16p (data not shown)
cofractionated with markers of
the Golgi (Fig.
7F, right, shows the
data for Sec14p; data for
guanosine diphosphatase not shown), plasma
membrane, mitochondria,
or vacuoles (data not shown). None of the
peroxisomal matrix proteins
tested, including THI (Fig.
7E, second
part), AOX (Fig.
7E, third
part), and ICL (data not shown), was
associated with the ER at
any time. These data confirm that the transit
of matrix proteins
to the peroxisome does not occur via the
ER.
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FIG. 7.
The sec238A mutation significantly delays,
but does not prevent, the exit of Pex2p from the ER and differentially
affects the peroxisomal import of Pex2p, THI, and AOX. (A to D)
The sec238A mutant strain grown in YPBO at 32°C was
pulse-labeled for 3 min with
L-[35S]methionine and chased with unlabeled
L-methionine. Samples were taken at the
indicated chase times. Cells were subjected to subcellular
fractionation, and the 20KgP fractions were further fractionated
by isopycnic centrifugation on
discontinuous sucrose density gradients. Pex2p, THI, AOX, Kar2p, and
Sec14p were immunoprecipitated from gradient fractions.
Immunoprecipitates were resolved by SDS-PAGE and visualized by
fluorography. Fractions containing either the complex formed between
peroxisomes and the ER or the Golgi alone (as judged from the
distributions of protein markers) were combined and treated with 30 mM
EDTA under conditions allowing complex disassembly. Treated samples
were fractionated by isopycnic centrifugation on discontinuous sucrose
density gradients. Pex2p, THI, AOX, Kar2p, and Sec14p were
immunoprecipitated from gradient fractions. Immunoprecipitates were
resolved by SDS-PAGE and visualized by fluorography. (E and F) The
sec238A mutant strain was grown in YPBO at 32°C, shifted
to 22°C for 2 h, pulse-labeled at 22°C for 3 min with
L-[35S]methionine, and chased with unlabeled
L-methionine. Samples were taken at the indicated chase
times. Cells were subjected to subcellular fractionation, and the 20KgP
fractions were further fractionated by isopycnic centrifugation on
discontinuous sucrose density gradients. Pex2p, THI, AOX, Kar2p, and
Sec14p were immunoprecipitated from gradient fractions.
Immunoprecipitates were resolved by SDS-PAGE and visualized by
fluorography. Peak fractions in which markers of peroxisomes (P),
the plasma membrane (PM), the ER, and the Golgi (G) localized are
indicated. (A and E) sec238A mutant cells taken at chase
time 0 at 32°C (A) or 22°C (E) were also subjected to subcellular
fractionation to yield 20KgP, 245KgP, and 245KgS (cytosolic) fractions.
Pex2p was immunoprecipitated from different subcellular fractions, and
immunoprecipitates were resolved by SDS-PAGE and visualized by
fluorography. (G) Fluorograms in panels B and E (left side) were
quantitated by densitometry. The levels of ER-associated Pex2p in
sec238A mutant cells grown at 22 or 32°C at the indicated
chase times are relative to the level of ER-associated Pex2p in
sec238A mutant cells grown at 22°C at chase time 0, which
was set at 100%. The half-times (t1/2) for the
exit of Pex2p from the ER for the sec238A mutant strain
grown at 22 or 32°C were calculated. (H) Fluorograms in panel B and
of the first three parts of panel E were quantitated by densitometry.
The levels of Pex2p, THI, and AOX imported into peroxisomes in
sec238A mutant cells grown at 22 or 32°C at the indicated
times postchase are relative to the levels of the corresponding
proteins imported into peroxisomes of the sec238A mutant
strain grown at 22°C after a 60-min chase, which were set at 100%.
The half-times for the import of Pex2p, THI, and AOX into peroxisomes
of the sec238A mutant strain grown at 22 or 32°C were
calculated.
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In
sec238A mutant cells grown at 32°C, the temperature
restrictive for protein secretion, the peroxisomes
pelletable at low
speed (20,000 ×
gmax)
form a complex with the ER in vitro (
47).
Due to the
formation of this complex, peroxisomal matrix proteins
(Fig.
7A shows
the data for THI and AOX; data for ICL not shown)
and ER marker
proteins (Fig.
7C, left, shows the data for Kar2p;
data for Sls1p and
NADPH:cytochrome
c reductase not shown) peaked
in fraction
16 of a discontinuous sucrose density gradient of
the 20KgP subcellular
fraction. While the distribution of peroxisomal
proteins around
fraction 16 coincided with the distribution of
ER luminal and membrane
proteins, their distribution did not coincide
with the distribution of
markers for the Golgi apparatus (Fig.
7C, right, shows the data for
Sec14p; data for guanosine diphosphatase
not shown),
mitochondria, vacuoles, or plasma membrane (data not
shown). Peroxisomes (Fig.
7B) could be separated from the ER (Fig.
7D)
in the complex by treatment with EDTA (
45). These
peroxisomes
were intact and essentially free from
contamination by other organelles,
as judged by protease protection,
flotation on a sucrose gradient,
and electron microscopy (data not
shown). By using these conditions
for disassembly, we demonstrated that
in
sec238A mutant cells
grown at the restrictive
temperature, the major portion of pulse-labeled,
unchased Pex2p (Fig.
7B, left panel) and Pex16p (data not shown)
localized to the ER. The
exit of Pex2p and Pex16p from the ER
was significantly delayed, but not
blocked, by the
sec238A mutation
at 32°C (Fig.
7B and G
show the data for Pex2p; data for Pex16p
not shown). The negative
effect of the
sec238A mutation at 32°C
on the exit of
Pex2p and Pex16p from the ER was much less pronounced
than that of the
pex1-1 and
pex6KO mutations (see above). However,
the rates of import of both Pex2p and Pex16p into
peroxisomes
were comparable in
sec238A mutant
cells grown at 32°C and in
pex1-1 and
pex6KO
mutant cells (half-times for the peroxisomal import
of both proteins
were >60 min in all of these strains grown at
the restrictive
temperature). Furthermore, while the
sec238A mutation
at
32°C severely affected the import of Pex2p (Fig.
7H), Pex16p
(data
not shown), and THI (Fig.
7H) into the peroxisome, its
effect
on the import of AOX (Fig.
7H) and ICL (data not shown) was much
less pronounced. Therefore, these data also suggest that the import
of
Pex2p, Pex16p, and THI into peroxisomes may occur by a
mechanism
distinct from that serving for the import of AOX and ICL.
Pex2p and Pex16p are subjected to N-linked core
glycosylation in the ER lumen and are delivered from
the ER to the peroxisome in glycosylated form.
Pex2p and Pex16p were immunoprecipitated from the ER or
peroxisomes of cells pulse-labeled with
radiolabeled methionine and chased with unlabeled methionine for 5 or
60 min, respectively. Immunoprecipitated Pex2p and
Pex16p were treated with endo H, which cleaves the bond between two
N-acetyl-D-glucosamine residues in the
core of N-linked oligosaccharides attached to the polypeptide backbone
(7). Treatment with endo H increased the
electrophoretic mobilities of both the ER- and
peroxisome-associated forms of Pex2p and Pex16p from
wild-type and mutant cells (Fig. 8). The difference in apparent molecular mass between endo H-treated and untreated polypeptides was approximately 2 kDa, which corresponds to the mass of a single core oligosaccharide chain (7). In contrast, endo H treatment of pulse-labeled and chased THI (mature form
in wild-type cells and precursor form in mutant cells), AOX, and ICL
immunoprecipitated from peroxisomes of wild-type and mutant strains did not change their electrophoretic mobilities (Fig. 8).
Therefore, these matrix proteins were not core glycosylated.
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FIG. 8.
Pex2p and Pex16p exist as glycosylated forms in the ER
and peroxisomes of wild-type (WT) and mutant strains. (A)
Wild-type and mutant cells grown in YPBO at 32°C were pulse-labeled
for 3 min with L-[35S]methionine and
subjected to either a 5- or 60-min chase with unlabeled
L-methionine, followed by immunoprecipitation and endo H
treatment of ER-associated Pex2p and Pex16p (5-min chase) or of
peroxisomal Pex2p, Pex16p, THI, AOX, and ICL (60-min chase). Cells were
subjected to subcellular fractionation, and the 20KgP fractions were
further fractionated by isopycnic centrifugation on discontinuous
sucrose density gradients. Pex2p and Pex16p were immunoprecipitated
from peak fractions in which protein markers of the ER or of
peroxisomes localized, while THI, AOX, and ICL were
subjected to immunoprecipitation from fractions in which peroxisomal
matrix proteins peaked (Fig. 6 and 7). Immunoprecipitates were divided
into two equal aliquots. One aliquot was digested with endo H (+),
while the second was mock digested (). Endo H-treated and untreated
proteins were resolved by SDS-PAGE and visualized by fluorography. (B)
Wild-type and mutant cells grown in YPBO at 32°C were separated into
two equal aliquots. Tunicamycin (+) (final concentration, 10 µg/ml)
was added to one aliquot 5 min before the addition of
L-[35S]methionine, while the second aliquot
was mock treated (). Cells from both aliquots were radiolabeled for
20 min with L-[35]methionine. Radiolabeling
was terminated by the addition of an equal volume of ice-cold 20 mM
NaN3 and 20 mM L-methionine. Pex2p and Pex16p
were immunoprecipitated from whole-cell lysates.
Immunoprecipitates were resolved by SDS-PAGE and visualized
by fluorography. The positions of the glycosylated (gPex2p and gPex16p)
and unglycosylated forms of Pex2p and Pex16p and of the precursor
(pTHI) and mature (mTHI) forms of THI are indicated at the right.
|
|
We also tested the effects of tunicamycin treatment of cells on the
glycosylation of Pex2p and Pex16p. Tunicamycin is a
specific
inhibitor of N-linked protein glycosylation in
vivo (
7). Wild-type
and mutant cells grown in YPBO at 32°C
were radiolabeled with
L-[
35S]methionine in
the presence or absence of tunicamycin. Treatment
of cells with
tunicamycin resulted in increased electrophoretic
mobilities of
radiolabeled Pex2p and Pex16p from wild-type and
mutant cells relative
to the mobilities of the two proteins from
untreated cells (Fig.
8B).
The difference in apparent molecular
mass between Pex2p and Pex16p from
tunicamycin-treated and untreated
cells was approximately 2 kDa and
similar to that observed between
endo H-treated and untreated forms of
both proteins, confirming
that both Pex2p and Pex16p are core
glycosylated in vivo and contain
a single core oligosaccharide chain.
We performed protease protection analysis of Pex2p and Pex16p to
analyze the topology of the N-linked core glycosylation
of
these proteins in both the ER and peroxisomes. In
wild-type
Y. lipolytica cells, Pex2p is an integral
peroxisomal membrane protein
that contains one predicted
membrane-spanning
-helix and one
canonical Asn-Xaa-Thr sequence for
N-linked glycosylation carboxyl
to this potential
transmembrane domain (
12). Treatment of the
ER and
peroxisomes from radiolabeled wild-type cells with trypsin
in the absence of detergent, followed by immunoprecipitation with
anti-Pex2p antibodies, revealed that a 21-kDa fragment of Pex2p
was
protected from degradation by trypsin in both the ER and
peroxisomes
(Fig.
9A, upper
right part). This 21-kDa fragment is core glycosylated
in both the ER
and peroxisomes and contains a single N-linked
oligosaccharide chain, as endo H treatment increased the
electrophoretic
mobilities of both the ER- and
peroxisome-associated forms of
this fragment, resulting in
the formation of a 19-kDa polypeptide
(Fig.
9B). An analysis of the
primary structure of Pex2p (
12)
shows that since (i) a
trypsin-sensitive cleavage site precedes
the potential
membrane-spanning
-helix of Pex2p, (ii) the only
canonical site for
N-linked glycosylation is carboxyl to this
transmembrane domain and is located towards the carboxyl terminus
of
Pex2p, and (iii) the predicted molecular mass (18.3 kDa) of
a fragment
of Pex2p that includes the membrane-spanning domain
and the amino acid
residues carboxyl to it is similar to the molecular
mass of the
identified protease-protected fragment of Pex2p after
endo H treatment
(19 kDa), we suggest that this carboxyl-terminal
part of Pex2p is
oriented towards the lumen of the ER and towards
the matrix of the
peroxisome. Therefore, N-linked core
glycosylation
of Pex2p occurs in the ER lumen, Pex2p is
delivered to the peroxisome
in glycosylated form, and the
transmembrane topology of Pex2p
is the same in the
peroxisome as in the ER.
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|
FIG. 9.
Orientations of Pex2p and Pex16p in the ER and in
peroxisomes and topology of N-linked core
glycosylation of Pex2p. (A) Wild-type cells grown in
YPBO at 32°C were pulse-labeled for 3 min with
L-[35S]methionine and subjected to either
a 5- or 60-min chase with unlabeled L-methionine for
immunoprecipitation and endo H treatment of the ER-associated (5-min
chase) or peroxisomal (60-min chase) forms of Pex2p and Pex16p. Cells
were subjected to subcellular fractionation, and the 20KgP fractions
were further fractionated by isopycnic centrifugation on discontinuous
sucrose density gradients. ER and peroxisomes purified as
described in the legend to Fig. 6 were incubated with 0, 4, 8, and 20 µg of trypsin in the presence (+) or absence () of 0.5% (vol/vol)
Triton X-100 for 40 min on ice. Reactions were terminated by addition
of SDS-PAGE sample buffer and boiling for 5 min. Reaction mixtures were
cooled, and Pex2p and Pex16p were immunoprecipitated.
Immunoprecipitates were resolved by SDS-PAGE and visualized by
fluorography. The positions of Pex2p and Pex16p and of the
glycosylated, trypsin-resistant fragment of Pex2p (21-kDa gt-Pex2p) are
indicated at the right. (B) Samples of the 21-kDa trypsin-resistant
fragment of Pex2p from both the ER and peroxisomes were
divided into two equal aliquots. One aliquot was digested with endo H
(+), while the second was mock digested (). Endo H-treated and
untreated proteins were resolved by SDS-PAGE and visualized by
fluorography. The positions of the glycosylated, trypsin-resistant
fragment of Pex2p (21-kDa gt-Pex2p) and the trypsin-resistant fragment
of Pex2p deglycosylated after endo H treatment (19-kDa t-Pex2p) are
indicated at the right. The values at the left are molecular mass
standards (in kilodaltons).
|
|
Pex16p is an intraperoxisomal peripheral membrane protein in wild-type
Y. lipolytica cells (
11). Treatment of the
ER and
peroxisomes of wild-type cells with trypsin in
the absence of
detergent, followed by immunoprecipitation of
radiolabeled Pex16p,
revealed that Pex16p is protected from
protease digestion in both
the ER and peroxisomes (Fig.
9A,
bottom right part). Therefore,
N-linked core
glycosylation of Pex16p occurs in the ER lumen and
Pex16p is targeted from the ER lumen to the peroxisomal matrix
in
glycosylated form.
| |
DISCUSSION |
Here we report that in wild-type Y. lipolytica,
trafficking of the peroxisomal membrane proteins Pex2p and Pex16p to
the peroxisome occurs via the ER and results in the
glycosylation of both proteins in the ER lumen but does
not involve their transit through the Golgi as an intermediate step. We
also demonstrate that mutations in the SEC238,
SRP54, PEX1, and PEX6 genes not only
cause defects in the exit of pAEP and other secretory proteins from the
ER and in protein secretion but also significantly delay or prevent the exit of Pex2p and Pex16p from the ER and affect the assembly of functionally intact peroxisomes. The SRP54 gene
encodes the Y. lipolytica homolog of the Srp54p
component of the signal recognition particle involved in protein
translocation across the ER membrane (24), while the
PEX1 and PEX6 genes encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like
ATPases that are essential not only for peroxisome
biogenesis but also for homo- and heterotypic fusion events required
for the assembly of other organelles (46). While previous
morphological studies of mammalian and yeast cells have shown a close
association between peroxisomes and the ER (for reviews,
see references 15 and 22) and
recent genetic and biochemical data have suggested a dual role for the
ER in supplying phospholipids for the formation of the
peroxisomal membrane (45) and in protein
trafficking to peroxisomes (2, 4, 13, 46, 50,
51), this study provides the first genetic evidence that the ER
is required for the assembly of functionally intact
peroxisomes in Y. lipolytica. A requirement for the ER in peroxisome assembly is also supported by our
data showing that not only Pex1p and Pex6p, but also other peroxins essential for peroxisome biogenesis in Y. lipolytica, are required for the exit of secretory proteins from
the ER and for their delivery to the extracellular medium
(46; this study).
The sec238A and srp54KO mutations at the
restrictive temperature of 32°C, and the pex1-1 and
pex6KO mutations at both 22 and 32°C, affected the export
of AEP to the extracellular medium and caused the accumulation of pAEP
in the ER. The rate and efficiency of secretion of all other secretory
proteins were affected in these mutants to similar extents. These data
suggest that in Y. lipolytica, Sec238p, Srp54p, Pex1p,
and Pex6p are essential for the exit of secretory proteins from the ER.
Unexpectedly, none of the sec238A, srp54KO,
pex1-1, or pex6KO mutations affected the
growth of Y. lipolytica in glucose-containing YEPD
medium, even at temperatures at which protein secretion was
impaired. In contrast, previous studies have shown that all
temperature-sensitive mutants of the yeast S. cerevisiae
affected in protein secretion are unable to grow in glucose-containing
media at temperatures restrictive for secretion (29, 30,
52). The observed differences in growth between the secretory
mutants of these two yeast species are probably due to essential
differences in the relationship between protein secretion and cell
surface growth in these yeasts. S. cerevisiae apparently has
one major pathway that is common for the secretion of proteins into the
cell envelope/extracellular medium and for the export of materials for
plasma membrane and cell wall synthesis (18, 28-31, 38).
Therefore, any mutational block in protein secretion also affects cell
surface growth. In contrast, our recent data have demonstrated that
protein secretion and cell surface growth in Y. lipolytica are served by distinct pathways (46).
Therefore, the sec238A, srp54KO,
pex1-1, and pex6KO mutations affect protein
secretion but do not compromise the export of plasma membrane and cell
wall-associated proteins during the yeast mode of growth of
Y. lipolytica in YEPD.
While neither the sec238A nor the srp54KO
mutation affected growth in YEPD, which does not require intact
peroxisomes, both mutations caused temperature-sensitive
growth in oleic acid-containing YPBO medium, the metabolism of which
requires the assembly of functionally intact peroxisomes.
Furthermore, the pex1-1 and pex6KO mutations
affected growth in YPBO at both 22 and 32°C but did not compromise
growth in YEPD at either temperature. Growth defects of these mutants
in YPBO were not caused by nonspecific (secondary) effects of the gene
mutations on cell viability and/or on the level of synthesis of
total phospholipid in YPBO (data not shown). Rather, the
combined data of electron and immunofluorescence microscopy and of
pulse-chase analysis demonstrated that Sec238p, Srp54p, Pex1p, and
Pex6p perform an essential and specific role in the import of matrix
and membrane proteins into the peroxisome. None of the
sec238A, srp54KO, pex1-1, or
pex6KO mutations compromised the synthesis of peroxisomal
proteins (data not shown). However, all of these mutations at the
restrictive temperature significantly reduced the size and number of
peroxisomes, caused the mislocalization of a large fraction
of peroxisomal matrix proteins and of some fraction of the peroxisomal
membrane proteins Pex2p and Pex16p to the cytosol, and selectively
affected the rates and efficiencies of import of individual peroxisomal
proteins into the organelle.
The combined data of immunofluorescence microscopy, pulse-chase
analysis, subcellular fractionation, protease protection, and endo H
digestion showed that in Y. lipolytica wild-type
cells, the trafficking of Pex2p and Pex16p to the
peroxisome occurs via the ER and results in the core
glycosylation of both proteins in the ER but does not
involve their transit through the Golgi. N-linked core
glycosylation of Pex2p and Pex16p occurs in the ER
lumen, and both proteins are delivered from the ER to the
peroxisome in glycosylated form. The transmembrane topology
of the integral membrane protein Pex2p in the ER and the peroxisomal
membrane is the same, with the carboxyl terminus of Pex2p oriented
towards the lumen of the ER and towards the matrix of the
peroxisome. Glycosylated Pex16p is localized within the ER
lumen and the peroxisomal matrix and is associated with the membranes
of both organelles. The structural features of Pex2p and Pex16p that
mediate their targeting from the cytosol to the ER, and from the ER to
the peroxisomal membrane, remain to be determined. Pex2p of
Y. lipolytica (11) contains a recently
identified peroxisomal targeting signal, termed mPTS, for peroxisomal
integral membrane proteins (9). One characteristic feature
of peroxisomal membrane proteins containing mPTS motifs, including
Pex2p of Y. lipolytica and Pmp47 of S. cerevisiae (9), is the presence of a canonical
Asn-Xaa-Thr sequence for N-linked glycosylation in
mPTS. Pex16p of Y. lipolytica also contains this canonical sequence (11). Our data demonstrate that both
Pex2p and Pex16p in Y. lipolytica are glycosylated in
the ER lumen and probably contain a single core N-linked
oligosaccharide chain. The importance of this
N-glycosylation for the targeting of Pex2p and Pex16p
to the ER and/or peroxisome is unknown. Interestingly, the
amino-terminal 16 amino acids preceding the mPTS of the
peroxisomal integral membrane protein, Pex3p, of the yeast
Hansenula polymorpha, while insufficient to target Pex3p to
peroxisomes, have been shown to be able to sort catalase,
lacking its carboxyl-terminal peroxisomal targeting signal 1, to the ER
and nuclear envelope (2). However, fusion of both the
amino-terminal 16 amino acids and mPTS of Pex3p to the truncated
catalase led to targeting of the fusion protein to the peroxisomal
membrane (2). We suspect that the targeting of some
peroxisomal membrane proteins, including Pex2p and Pex16p of
Y. lipolytica and Pex3p of H. polymorpha,
from the cytosol to the ER, and from the ER to the peroxisomal
membrane, can be mediated by distinct targeting signals. In contrast to
the transit of the peroxisomal membrane proteins Pex2p and Pex16p, the
trafficking of the peroxisomal matrix proteins THI, AOX, and ICL in
Y. lipolytica cells does not occur via the ER and does
not involve the glycosylation of these proteins.
In conclusion, the results described herein provide evidence for the
essential role of the ER in the assembly of functionally intact
peroxisomes. We show that in Y. lipolytica,
the delivery of Pex2p and Pex16p to the peroxisomal membrane occurs via
the ER, results in their glycosylation in the ER lumen,
does not involve their transit through the Golgi, and requires the
products of the SEC238, SRP54, PEX1,
and PEX6 genes. The molecular mechanisms by which Sec238p,
Srp54p, Pex1p, and Pex6p function in the exit of peroxisomal membrane
proteins from the ER and in peroxisomal protein import are currently
being investigated.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Natural Sciences and
Engineering Research Council of Canada to R.A.R. R.A.R. is a
Medical Research Council of Canada Senior Scientist and an
International Research Scholar of the Howard Hughes Medical Institute.
We thank Honey Chan for help with electron microscopy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell Biology and Anatomy, University of Alberta, Medical Sciences
Building 5-14, Edmonton, Alberta T6G 2H7, Canada. Phone: (403)
492-9868. Fax: (403) 492-9278. E-mail:
rrachubi{at}anat.med.ualberta.ca.
| |
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Mol Cell Biol, May 1998, p. 2789-2803, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
