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J. Biol. Chem., Vol. 277, Issue 25, 22395-22401, June 21, 2002
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From the Department of Molecular and Cell Biology, Howard Hughes
Medical Institute, University of California,
Berkeley, California 94720
Received for publication, January 15, 2002, and in revised form, April 9, 2002
The yeast plasma membrane
H+-ATPase Pma1p is one of the most abundant proteins
to traverse the secretory pathway. Newly synthesized Pma1p exits the
endoplasmic reticulum (ER) via COPII-coated vesicles bound for the
Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p
into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a
unique role for Lst1p in ER export. Vesicles formed with mixed
Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we
examined the relationship between Pma1p biosynthesis and the
requirement for this novel coat subunit. We show that Pma1p forms a
large oligomeric complex of >1 MDa in the ER, which is packaged into
COPII vesicles. Furthermore, oligomerization of Pma1p is linked to
membrane lipid composition; Pma1p is rendered monomeric in cells
depleted of ceramide, suggesting that association with lipid rafts may
influence oligomerization. Surprisingly, monomeric Pma1p present in
ceramide-deficient membranes can be exported from the ER in COPII
vesicles in a reaction that is stimulated by Lst1p. We suggest that
Lst1p directly conveys Pma1p into a COPII vesicle and that the larger
size of mixed Sec24pLst1p COPII vesicles is not essential to the
packaging of large oligomeric complexes.
An essential protein of the yeast plasma membrane is the
H+-ATPase Pma1p. At steady state, it composes >25% of the
total protein at the plasma membrane, where it generates a proton
gradient that maintains the intracellular pH and drives the import of
nutrients (1). Pma1p spans the lipid bilayer with 10 transmembrane
segments and belongs to the family of P2-type ATPases,
which includes the Na+,K+-ATPases and
Ca2+-ATPases of the mammalian plasma membrane (2,
3).
As with other integral membrane proteins destined for the plasma
membrane, Pma1p is translocated into the endoplasmic reticulum (ER)1 and travels through a
series of transport vesicles to its final destination (4, 5). En route,
Pma1p and the glycosylphosphatidylinositol-anchored protein
Gas1p become associated with lipid rafts (6, 7). Interestingly, raft
association of Gas1p initiates in the ER (6), unlike in mammalian
cells, where glycosylphosphatidylinositol-anchored proteins enter rafts
in post-ER compartments (8, 9). Exit out of the ER is mediated by COPII
(coat protein complex II)
vesicles, a universal mechanism in eukaryotes employing a set of
cytoplasmic coat proteins (10). Budding is regulated by the small
G-protein Sar1p, which recruits two complexes, Sec23p-Sec24p and
Sec13p-Sec31p (10-12). Assembly of these three components on the
surface of liposomes is sufficient to deform the lipid bilayer to
generate small coated vesicles (11). On the ER membrane, Sar1p and
Sec23p-Sec24p promote the capture of a number of cargo proteins,
suggesting that Sec23p-Sec24p may function to selectively engage cargo
proteins during coat assembly (13, 14).
Homologs of Sec24p have been identified in yeast and mammals (15, 16)
and may act to diversify the range of cargo proteins recruited into a
nascent vesicle. In yeast, the Sec24p homolog Lst1p was discovered in a
screen for synthetic interactions with the sec13-1 allele
(lethal with
sec-thirteen)
(15). A null mutant of lst1 is viable, although sensitive to
low pH resulting from a reduced flux of Pma1p out of the ER (15).
Efficient incorporation of Pma1p into COPII vesicles requires a
combination of both Sec24p and Lst1p complexes, unlike other cargo
proteins studied to date (16). In addition to the enhancement of Pma1p packaging, Lst1p-positive vesicles are ~15% larger than vesicles generated with standard COPII subunits (17).
In this study, we investigated the role of Pma1p oligomerization in
Lst1p-dependent Pma1p transport. Both oligomeric and
monomeric Pma1p are packaged into COPII vesicles, and both forms of
Pma1p require Lst1p for efficient transport from the ER.
Reagents, Strains, and Plasmids--
Reagents were purchased
from Sigma unless otherwise noted. Aureobasidin A was
purchased from Takara Bio Inc. (Shiga, Japan). Rich medium
(yeast extract/peptone/dextrose) and minimal medium containing
either 2% glucose (synthetic dextrose medium) or 2% raffinose were prepared as described (17). The yeast strains used in this study were as follows: YPH499 (MATa
ade2-101oc his3- Preparation of Proteins and Cellular Components--
Microsomal
membranes were prepared as described (19) unless otherwise noted. Both
35S-labeled and unlabeled cells were converted to
semi-intact spheroplasts as described (17). Purification of
Sec23p-Lst1p (17) and Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p (20) is
described elsewhere.
Blue Native (BN)-PAGE--
Membranes were solubilized with
detergents at 4 °C (except SDS at room temperature) for 30 min in 50 mM Tris-HCl, 150 mM NaCl, and 1 mM
EDTA, pH 7.5, and then centrifuged for 5 min at 15,000 × g. BN-PAGE sample buffer (0.1 volume of 5% Coomassie
Brilliant Blue G-250, 50 mM BisTris, 750 mM 6-aminocaproic acid, and 50% glycerol, pH 7.0) was
added to the supernatant, which was separated on 4-10% polyacrylamide
gradient gels as described (21) with high molecular mass markers from
Amersham Biosciences.
Pulse-Chase Kinetics of Pma1p Stability--
Cells grown
overnight at 24 °C in yeast extract/peptone/dextrose medium were
harvested, washed twice, and resuspended in synthetic dextrose
medium at ~1 A600/ml. After 15 min at
30 °C, cells were pulsed with 60 µCi of
35S-Promix/A600 (Amersham
Biosciences) for 3 min. Chase was initiated by addition of 50 mM methionine, 10 mM cysteine, 4% yeast
extract, and 2% dextrose. At each time point, cells (1 A600 unit) were removed to ice and arrested with
20 mM NaN3/KF. Cells converted to spheroplasts
were lysed with 1% SDS at 55 °C for 5 min, and lysates were diluted
with 1 ml of immunoprecipitation buffer (15 mM
Tris-HCl, 1% Triton X-100, 150 mM NaCl, and 0.05% SDS, pH
7.5) and centrifuged for 5 min at 15,000 × g, and the
supernatant was incubated with anti-HA antibodies (HA.11, Berkeley
Antibody Co., Berkeley, CA) and protein G-Sepharose (Amersham
Biosciences). Immunoprecipitates were analyzed by SDS-PAGE and with a
PhosphorImager using ImageQuant software (Molecular Dynamics, Inc.,
Sunnyvale, CA).
Detergent-insoluble Glycolipid-enriched Complex (DIG)
Isolation--
Microsomes and spheroplasts were centrifuged at
15,000 × g for 5 min, and vesicles were centrifuged at
100,000 × g for 20 min and resuspended in buffer A
(1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl,
5 mM EDTA, and 1 mM phenylmethylsulfonyl
fluoride, pH 7.4) at 4 °C for 30 min. Extracts were centrifuged at
1000 × g for 5 min, and the supernatant (330 µl) was
adjusted to 35% Optiprep (Nycomed, Oslo, Norway) with 770 µl of 50%
Optiprep in buffer A, overlaid with 1.4 ml of 30% Optiprep in buffer A
and 0.5 ml of buffer A, and centrifuged at 4 °C for 16 h at
40,000 rpm in a Beckman SW 55 rotor. Fractions (200 µl) were analyzed by either immunoblotting or immunoprecipitation. The indicated fractions (400 µl) were immunoprecipitated by addition of SDS to 1%
at 55 °C for 5 min and dilution with 4 ml of immunoprecipitation buffer; immunoprecipitations were performed sequentially using anti-HA,
anti-Gas1p, and anti-Sec22p antisera.
ER Vesicle Budding Assay--
Vesicle budding from microsomal
membranes and semi-intact cells was performed as described (17, 20).
Membranes were first washed with buffer B (20 mM Hepes, 250 mM sorbitol, 150 mM KOAc, and 5 mM
Mg(OAc)2, pH 6.8); and unless otherwise noted, budding reactions contained 20 µg/ml Sar1p and Sec13p-Sec31p and either 20 µg/ml Sec23p-Sec24p or 10 µg/ml each Sec23p-Sec24p and Sec23p-Lst1p as well as the non-hydrolyzable GTP analog GMP-PMP (Roche Molecular Biochemicals) and an ATP regeneration system. Budding reactions for
BN-PAGE used 100 µg of microsomes (125-µl reaction), and those for
Fig. 3B used 400 µg of microsomes (500-µl reaction).
Budding from labeled semi-intact cells used cells at
A600 = 1.25 (125-µl reaction). For electron
microscopy, vesicles were generated from 1.5 mg of microsomes washed to
remove endogenous COPII proteins (2× buffer B + 2.5 M
urea, 2× buffer B + 1 mM GTP, and 3× buffer B). All
reactions were performed for 30 min at 25 °C.
Electron Microscopy--
The vesicle-enriched medium speed
supernatant (MSS) was centrifuged at 100,000 × g for
20 min. The vesicle pellet was processed as described (12). Vesicle
diameter was measured from scanned negatives (×30,000) using the
Photoshop measuring tool. A sample of 500 vesicles was measured for
each condition.
Pma1p Forms a Large Oligomeric Complex--
We used BN-PAGE to
examine the oligomeric state of Pma1p in microsomal membranes.
Membranes generated from a strain expressing a chromosomal HA-tagged
form of Pma1p were solubilized with different detergents, and the
proteins were separated by BN-PAGE (Fig.
1A). Pma1p migrated as a
distinct high molecular mass complex of ~1.8 MDa in extracts
solubilized with Triton X-100, digitonin, or n-dodecyl maltoside. Pma1p solubilized with SDS migrated with an apparent molecular mass of 160 kDa, most likely corresponding to monomeric protein, which has a predicted molecular mass of ~100 kDa.
Interestingly, two bands of intermediate size were observed in
n-dodecyl maltoside extracts; the apparent sizes of these
intermediates corresponded closely to those of predicted trimeric
and hexameric complexes of Pma1p (Fig. 1B) and may represent
partial dissociation of a more abundant dodecameric species. Although
the size of the 1.8-MDa species is suggestive of a homo-oligomeric
complex of 12 Pma1p molecules (possibly arranged as four trimers), we
cannot yet rule out the presence of additional proteins in the complex.
To determine whether the Pma1p multimer originates in the ER, we
examined newly synthesized Pma1p in the sec18-1 strain,
which is rapidly blocked in ER-to-Golgi transport at 37 °C. Cells
were shifted to 37 °C for 5 min, and expression of epitope-tagged
Pma1p was induced with galactose for 30 min. Oligomerization of the induced Pma1p was observed in both wild-type and sec18-1
extracts (Fig. 1C), whereas no protein was detected in the
absence of galactose (data not shown). Although Pma1p in wild-type
cells was found predominantly as the oligomeric species, imposition of
a sec18 block resulted in approximately equal amounts
of oligomeric and monomeric Pma1p. Thus, Pma1p can oligomerize in the
ER, but oligomerization is somewhat impeded by a block in ER-to-Golgi transport.
Oligomerization Is Dependent on the Ceramide Biosynthesis
Pathway--
The observation that Pma1p associates with DIGs or lipid
rafts (6) prompted us to investigate whether oligomerization is correlated with sphingolipid biosynthesis. To deplete cell membranes of
sphingolipids, we used the lcb1-100 strain, which has a
temperature-sensitive mutation in a subunit of serine
palmitoyltransferase and is unable to synthesize sphingoid base,
ceramide, and sphingolipids (22). Bagnat et al. (6) found
that Pma1p isolated from lcb1-100 cells grown at restrictive
temperature is no longer raft-associated. We examined the oligomeric
state of Pma1p in wild-type and lcb1-100 cells grown either
at 24 °C or after a 2-h shift to 37 °C. Unlike wild-type
membranes, Pma1p isolated from lcb1-100 cells migrated almost exclusively as the monomeric form, regardless of the temperature at which the cells were grown (Fig.
2A). This is consistent with the observation that sphingolipid levels in lcb1-100 cells
are considerably lower than those in wild-type cells even at 24 °C (23).
We investigated whether addition of exogenous sphingoid base in
the form of phytosphingosine (PHS) could restore the levels of
sphingolipid sufficiently to allow oligomerization of Pma1p. Both
wild-type and lcb1-100 cells grown at 24 °C were
supplemented with various levels of PHS for 2 h and analyzed by
BN-PAGE (Fig. 2B). Addition of 1-10 µM PHS to
lcb1-100 cells promoted increased oligomerization of Pma1p.
Interestingly, the proportion of oligomeric Pma1p in wild-type cells
was also increased by addition of low levels of PHS, suggesting that
oligomerization is intimately linked with sphingolipid levels in the cell.
Depletion of sphingolipid levels can also be achieved by addition of
drugs such as myriocin, which inhibits serine palmitoyltransferase activity (24), and aureobasidin A (AbA), which blocks synthesis of the
sphingolipid inositol phosphorylceramide from ceramide (25). To
determine whether myriocin influences Pma1p oligomerization, we treated
cells with up to 40 µg/ml myriocin for 2 h. Under these conditions, we were unable to observe a significant shift of the steady-state levels of oligomeric Pma1p to monomer (data not shown), suggesting either that depletion of sphingolipid was not sufficient during the 2-h treatment or that, once formed, Pma1p oligomers are
resistant to a decrease in sphingolipid levels. To analyze the effects
of myriocin and AbA on newly synthesized Pma1p, we used the
galactose-inducible HA-tagged Pma1p plasmid described above. Wild-type
cells expressing endogenous untagged Pma1p were grown in non-inducing
medium at 24 °C and treated with myriocin or AbA for 15 min before
induction of HA-tagged Pma1p. Unlike control cells, myriocin-treated
cells showed a defect in the ability of newly synthesized Pma1p to
oligomerize (Fig. 2C). However, oligomerization was not
prevented in cells treated with AbA (Fig. 2C), suggesting
that either ceramide or sphingoid base (but not sphingolipids per
se) is required for Pma1p oligomerization. Under these conditions,
AbA prevented the incorporation of
12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))ceramide into inositol phosphorylceramide as determined by TLC
(26).2
We assessed the effect of ceramide depletion on the stability of Pma1p
by a 35S pulse-chase analysis. In wild-type cells, Pma1p
remained stable throughout the chase period (Fig. 2D),
consistent with its long half-life (5). In both lcb1-100
cells and myriocin-treated wild-type cells, however, Pma1p was markedly
destabilized. Myriocin failed to induce degradation of Pma1p in
pep4 Lipid Raft Association in the Early Secretory Pathway--
A block
in ceramide synthesis by myriocin treatment or by growth of
lcb1-100 cells at nonpermissive temperature prevents the ER-to-Golgi transport of the glycosylphosphatidylinositol-anchored protein Gas1p (24, 27), which associates with DIGs before leaving the
ER (6). To determine whether oligomerization of Pma1p in the ER
coincides with its entry into Triton X-100-insoluble DIGs, we isolated
DIGs from pulse-labeled sec18-1 cells.
At permissive temperature, the proportion of Pma1p associated
with DIGs increased markedly after prolonged chase (Fig.
3A). Gas1p displayed a similar
pattern, with a proportion of both the ER form (2 min) and the
Golgi-modified form (15 min) in the detergent-insoluble fraction (Fig.
3A). At 37 °C, the pool of DIG-associated Gas1p increased
after extended chase, whereas Pma1p was no longer observed in DIGs
(Fig. 3A). This suggests that, unlike Gas1p, Pma1p does not
associate with lipid rafts in the ER or, alternatively, that a block in
ER export diminishes the stability of the association of Pma1p with
lipid rafts.
To distinguish between these possibilities, we analyzed the detergent
solubility of Pma1p in ER-derived COPII vesicles. Microsomal membranes
were used in a vesicle budding reaction using purified COPII proteins
(Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p) together with the Sec24p
homolog Lst1p. The vesicle-enriched MSS was subjected to centrifugation
at 100,000 × g to pellet vesicles, from which DIGs
were isolated by flotation as described above. Vesicles generated in
the presence of nucleotide showed efficient budding of Pma1p, whereas
the ER resident Sec61p was not packaged into vesicles (Fig.
3B). A significant proportion of Pma1p in COPII vesicles was
in a detergent-insoluble pool (fractions 5 and 6) (Fig. 3B). In contrast, the SNARE proteins Sec22p, Bet1p, and Bos1p were efficiently incorporated into COPII vesicles, but were not
DIG-associated (Fig. 3B and data not shown).
To confirm that the DIG-associated Pma1p observed in the in
vitro budding reaction corresponded to newly synthesized protein from the ER, we performed a budding reaction using semi-intact cells
generated after a brief period of radiolabeling. Vesicle DIGs were
isolated by flotation, and Pma1p was immunoprecipitated from fractions
representative of detergent-insoluble and detergent-soluble material. A
significant fraction (11%) of Pma1p in COPII vesicles was
DIG-associated (Fig. 3C). Thus, Pma1p association with DIGs either is stabilized by entry into COPII vesicles or occurs initially in the ER, but is disrupted by the imposition of a secretion block.
Oligomeric Pma1p Is Packaged into COPII Vesicles--
The
dependence of Pma1p oligomerization on ceramide biosynthesis, together
with the observation that Pma1p is associated with DIGs in COPII
vesicles, prompted us to investigate the effect of ceramide depletion
on the oligomeric state of Pma1p packaged into vesicles. COPII vesicles
were generated from wild-type microsomes or microsomes derived from
lcb1-100 cells and analyzed by BN-PAGE. In vesicles
generated from wild-type microsomes, Pma1p was packaged as the large
oligomeric species (Fig. 4A).
Interestingly, COPII vesicles generated from lcb1-100
microsomes packaged Pma1p, but the protein remained in the monomeric
form (Fig. 4A).
We investigated whether oligomerization might enhance the uptake of
Pma1p into vesicles, possibly by allowing more efficient recruitment of
vesicle coat components to the ER membrane. We performed budding
reactions with 35S-labeled cells grown in the presence of
myriocin for 2 h, a treatment that renders Pma1p monomeric (see
Fig. 2C). lst1-null cells were used to allow an
accurate titration of Pma1p budding by addition of increasing amounts
of purified Lst1p and Sec24p complexes in the presence of constant
levels of Sar1p and Sec13p-Sec31p. In both myriocin-treated and control
membranes, Pma1p budding was stimulated by increasing concentrations of
Sec24p and Lst1p complexes (Fig. 4B). No significant
differences in budding efficiency were observed between wild-type and
myriocin-treated membranes, regardless of the concentration of COPII
components provided.
Lst1p Stimulates the Packaging of Both Oligomeric and Monomeric
Pma1p--
We next investigated more directly the relationship
between the oligomeric state of Pma1p and the requirement for Lst1p in ER export. Shimoni et al. (17) proposed two possible
mechanisms for the Lst1p-mediated stimulation of Pma1p budding. Lst1p
may act as a "cargo-adapter," binding directly to Pma1p to
efficiently recruit it into a nascent vesicle. Alternatively, Lst1p may
form larger vesicles with a lower membrane curvature and greater
surface area, thus indirectly facilitating the packaging of oligomeric Pma1p. If so, the ability of high levels of Sec24p to overcome the
requirement for Lst1p in Pma1p packaging (17) may reflect an increase
in the incorporation of monomeric Pma1p into nascent vesicles. To
address this issue, we compared the oligomeric state of Pma1p in
vesicles that had been generated with either a combination of Sec24p
and Lst1p complexes or with high levels of the Sec24p complex alone
(Fig. 4C). Analysis of vesicles by BN-PAGE revealed that the
presence of Lst1p was not essential for packaging oligomeric Pma1p,
which could be accommodated in Lst1p-negative vesicles given a
sufficient quantity of coat proteins (Fig. 4C).
We next considered whether Pma1p rendered monomeric by ceramide
depletion may bypass the requirement for Lst1p and be incorporated into
the smaller vesicles generated with the standard COPII proteins. Membranes were isolated from lst1 Packaging of Large Pma1p Complexes Does Not Determine Vesicle
Size--
The requirement for Lst1p to package both oligomeric and
monomeric Pma1p suggests a function as a cargo-specific adapter, rather
than an indirect role in accommodating large complexes. We considered
the possibility that the larger size of COPII vesicles produced with
the Lst1p complex was influenced by the incorporation of large
DIG-associated Pma1p oligomers. Pma1p is a particularly abundant
protein; thus, it is conceivable that its presence alone in
Lst1p-positive vesicles may influence vesicle size.
Large-scale budding reactions were performed using either
wild-type or lcb1-100 microsomes prepared from cells grown
at 24 °C and shifted to 37 °C for 1 h. Under these
conditions, Pma1p appeared monomeric in lcb1-100 membranes
(see Fig. 2A). Microsomal membranes were washed to remove
endogenous COPII components and then supplied with Sar1p,
Sec13p-Sec31p, and either the Sec24p complex alone or a mixture of both
Sec24p and Lst1p complexes, in addition to the non-hydrolyzable GTP
analog GMP-PMP to preserve vesicle coats. The high speed vesicle pellet
was analyzed by electron microscopy.
Vesicles with a discernible coat were generated under all conditions,
and no obvious differences in coat morphology were detected (data not
shown). Lst1p-positive vesicles produced from wild-type and
lcb1-100 membranes were of comparable size and were
~10-15% larger in diameter than Lst1p-negative vesicles (Table
I). Thus, incorporation of large Pma1p
complexes into Lst1p-positive COPII vesicles does not explicitly
determine the size of the vesicle produced.
Pma1p is a particularly abundant secretory protein that uniquely
requires Lst1p for packaging into COPII vesicles for ER export. We have
investigated the nature of this requirement with respect to the
quaternary structure of Pma1p. We found that, upon BN-PAGE, Pma1p
migrated as an oligomeric complex. Our estimate that this complex
represents a homododecamer of Pma1p is consistent with observations in
related yeast suggesting that H+-ATPases form higher order
structures. Formation of a multimer of 8-10 monomers was reported for
the Schizosaccharomyces pombe H+-ATPase (28),
whereas the Neurospora crassa H+-ATPase was
observed as a hexamer upon gradient centrifugation and crystallization
(29, 30). Oligomerization does not appear to be essential for activity,
however, as monomeric protein isolated from N. crassa was
reconstituted into proteoliposomes as fully active enzyme (31).
Recently, Bagnat et al. (7) reported that a fraction of
Saccharomyces cerevisiae Pma1p behaves as an oligomer of
~400 kDa on SDS/Triton X-100 velocity gradients. In contrast, we
observed by BN-PAGE that the majority of Pma1p migrated as an
~1.8-MDa complex under a variety of detergent conditions. However, a
small proportion of Pma1p migrated at ~490 kDa when membranes were
solubilized with SDS (Fig. 1A), which may correspond to the
complex observed by Bagnat et al. (7). Interestingly,
overexpression of a peripheral membrane protein (Ast1p) was found to
substantially increase the proportion of the ~400-kDa Pma1p complex,
in addition to conferring increased raft association on a mutant Pma1p
protein (7). Upon BN-PAGE, Ast1p did not co-migrate with the 1.8-MDa
Pma1p complex and thus does not contribute to the increased size of the
Pma1p oligomer we observed (data not shown). The association of Pma1p with lipid rafts and with Ast1p was proposed by Bagnat et
al. (7) to occur in the Golgi; however, our observation that
raft-associated Pma1p oligomers can be isolated from COPII vesicles
suggests that entry into rafts and oligomerization begin before arrival
at the Golgi.
The formation of an oligomeric Pma1p complex in the ER is consistent
with the observation that expression of a number of dominant-negative mutants of Pma1p results in the retention of both the mutant and wild-type proteins in the ER (18, 32). Formation of mixed oligomers
between the normal and mutant proteins could lead to the recognition of
the entire complex as misfolded and subject to ER quality control. This
model is supported by our observations that assembly of the multimeric
Pma1p complex is dependent on the level of ceramide biosynthesis and
that a block in this pathway relieves the dominant lethal phenotype of
a Pma1p mutant.3
The association of Pma1p with DIGs suggests one possible
mechanism for oligomerization whereby newly synthesized Pma1p
preferentially partitions into lipid microdomains, creating a local
concentration of protein sufficient to drive oligomerization. A
decrease in the flux of ceramide through the ER may result in the
fragmentation of these microdomains such that oligomerization is
prevented. Alternatively, oligomerization may proceed concurrently but
independently of the formation of nascent lipid rafts, requiring
only a product of the ceramide biosynthesis pathway. We are currently
examining the oligomeric state of Pma1p in membranes depleted of ergosterol.
In contrast with the transport of Gas1p, which fails to reach the Golgi
if ceramide biosynthesis is interrupted (24, 27), ER export of Pma1p is
not dependent on the presence of ceramide or on its oligomeric state.
However, the stability of Pma1p is dramatically altered by a disruption
in the ceramide biosynthesis pathway, resulting in a rerouting to the
vacuole for degradation.
A consequence of the formation of large oligomeric cargo such as Pma1p
may be the requirement for a more spacious transport vehicle (33). The
observation that oligomeric Pma1p is packaged into COPII vesicles has
allowed us to address the unique requirement for Lst1p in this process.
Our finding that efficient export of Pma1p requires Lst1p regardless of
cargo size suggests, however, that Lst1p functions to actively promote
Pma1p packaging, possibly by direct recruitment of Pma1p during
polymerization of the nascent vesicle coat. Once in COPII vesicles,
Pma1p can be co-immunoprecipitated with both the Lst1p and Sec24p
complexes (17); however, to confirm a direct interaction, it will be
necessary to reconstitute these associations in purified
proteoliposomes. Such a system may help to explain the contribution of
unique coat components such as Lst1p to vesicle formation. Our
observation that Lst1p generates larger vesicles from membranes
depleted of DIGs and oligomeric Pma1p and potentially other
sphingolipid-dependent protein complexes suggests that the
size of COPII vesicles is not driven by the nature of their cargo, but
by the intrinsic properties of their coats.
We thank members of the Schekman laboratory,
in particular E. Miller and F. Portillo, for generous help. Special
thanks to C. Chan and R. Lesch for preparation of reagents. We are
grateful to A. Chang and P. Malkus for communicating unpublished
results and H. Riezman for providing yeast strains.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 510-642-5686;
Fax: 510-642-7846; E-mail: schekman@uclink4.berkeley.edu.
Published, JBC Papers in Press, April 11, 2002, DOI 10.1074/jbc.M200450200
2
P. Malkus and R. Schekman, personal communication.
3
Q. Wang and A. Chang, personal communication.
The abbreviations used are:
ER, endoplasmic
reticulum;
HA, hemagglutinin;
BN, blue native;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
DIG, detergent-insoluble glycolipid-enriched complex;
GMP-PNP, guanyl-5'-yl imidodiphosphate;
MSS, medium speed supernatant;
PHS, phytosphingosine;
AbA, aureobasidin A;
SNARE, soluble NSF
attachment protein receptor.
Ceramide Biosynthesis Is Required for the Formation of the
Oligomeric H+-ATPase Pma1p in the Yeast Endoplasmic
Reticulum*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
200 leu2-1 lys2-801am trp1-63
ura3-52), RSY1578 (YPH499 pma1::HA-PMA1::LEU2),
MLY1 (MAT
lcb1-100 leu2 ura3 his4
pma1::HA-PMA1::LEU2) (HR2607, H. Riezman, University of Basel, Basel, Switzerland), RSY372
(MAT ura3-52 leu2-3,112 sec18-1), MLY18
(RSY372
pma1::HA-PMA1::LEU2), MLY4 (MAT ade2-1 his3-11,15 leu2-3,112 trp1-1
ura3-1 pep4::TRP1 pma1::HA-PMA1::LEU2),
and RSY1801 (RSY1578 lst1::HIS3).
Galactose induction of hemagglutinin (HA)-tagged Pma1p carried out
with pFP302 (18) in either YPH499 or RSY372.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pma1p forms an oligomeric complex in the ER.
A, microsomal membranes isolated from a strain expressing
HA-tagged Pma1p were solubilized with 1% Triton X-100, 1% digitonin
(DIG.), 0. 2 mg/ml n-dodecyl maltoside
(DDM), 2% octyl glucoside (OG) (25 µg of
microsomes), or 0.5% SDS (5 µg of microsomes). Protein extracts were
separated by BN-PAGE and immunoblotted with HA-specific antibodies.
B, the relative mobilities of molecular mass markers (bovine
serum albumin (67 kDa), lactate dehydrogenase (140 kDa), catalase (230 kDa), ferritin (440 kDa), and thyroglobulin (670 kDa)) were used
to size Pma1p complexes isolated from microsomes solubilized with
n-dodecyl maltoside. The protein band of lowest molecular
mass was designated as monomer (n = 1). C,
HA-Pma1p was induced by galactose for 30 min after wild-type
(WT) or sec18-1 cells were shifted to 37 °C
for 5 min. Cells were converted to spheroplasts, and proteins were
solubilized with Triton X-100 and analyzed by BN-PAGE and
immunoblotting with HA-specific antibodies. Newly synthesized
oligomeric Pma1p was observed in both wild-type and
sec18-blocked cells.
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Fig. 2.
Oligomerization of Pma1p is dependent on
ceramide synthesis. A, wild-type (WT; RSY1578)
and lcb1-100 microsomal membranes were solubilized with
Triton X-100 and analyzed by BN-PAGE and immunoblotting. Membranes were
isolated from either wild-type or lcb1-100 cells grown
either at 24 °C or after a 2-h shift to 37 °C. B,
addition of exogenous PHS restored oligomer assembly in
lcb1-100 cells. Wild-type and lcb1-100 cells
grown at 24 °C were supplemented with 0, 1, 2.5, and 10 µM PHS for 2 h before harvesting. Cells were
converted to spheroplasts, solubilized with Triton X-100, and analyzed
by BN-PAGE and immunoblotting. C, wild-type cells
(YPH499(pFP302)) grown in raffinose medium were treated for 15 min with
10 µg/ml myriocin, 10 µg/ml AbA, or ethanol only
(control) before galactose induction of HA-Pma1p. An
aliquot of cells (2 A units) was removed at 0 and 30 min after induction and converted to spheroplasts, and HA-Pma1p was
analyzed by BN-PAGE and immunoblotting. D, depletion of
ceramide decreased the stability of Pma1p. Cells were
pulse-radiolabeled for 3 min, followed by a chase period of 0-60 min
at 30 °C. HA-Pma1p was immunoprecipitated from cells at 1 A unit/time point. Wild-type and lcb1-100 cells
were grown at 24 °C and shifted to 30 °C for 15 min before
labeling. Myriocin treatment of wild-type (WT + myr.) and
pep4 (pep4 + myr.) cells was for 2 h
at 30 °C before labeling. The stability of Pma1p was quantified
relative to the amount present at 0 min.
cells, suggesting that monomeric Pma1p is permitted
to exit the ER, but is subsequently rerouted to the vacuole.
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Fig. 3.
Association of Pma1p with lipid rafts in the
ER and COPII vesicles. A, DIG-associated proteins were
isolated from sec18-1 strain MLY18 at permissive and
nonpermissive temperatures. Cells were pulse-labeled for 3 min either
at 24 °C or after pretreatment at 37 °C for 5 min, followed by a
2- or 15-min chase. Cells (2.5 A units) were converted to
spheroplasts and gently lysed with Triton X-100, and DIGs were isolated
by flotation. Representative gradient fractions of detergent-insoluble
(I) and detergent-soluble (S) material were
immunoprecipitated for Pma1p and Gas1p, and DIG-associated protein was
quantified relative to the total (T; 1:10). ER
(p) and Golgi (m) forms of Gas1p were quantitated
together. B, DIGs were isolated from COPII vesicles
generated from microsomal membranes (RSY1578) with Sar1p, Sec24p and
Lst1p complexes, and Sec13p-Sec31p in the presence (+) or absence ( 
)
of nucleotide. A fraction of the total reaction mixture (T;
1:100) and the MSS (+ and ; 1:40) was resolved by SDS-PAGE and
immunoblotted for Pma1p, Sec22p, and Sec61p. The remaining MSS was
solubilized with Triton X-100 and applied to an Optiprep gradient.
Fractions (200 µl) across the entire gradient were collected, and 10 µl of each was resolved by SDS-PAGE and immunoblotted for Pma1p and
Sec22p. A sizable proportion of Pma1p was found in the
detergent-insoluble fraction (I). C, cells
(RSY1578) were pulse-labeled for 3 min and converted to permeabilized
spheroplasts. Budding reactions (cells at 5 A units) were
performed as described for B. A fraction of the total
reaction (T; 1:50) and MSS (+ or nucleotide; 1:10) was
used to immunoprecipitate HA-Pma1p or Sec22p, and the budding
efficiency was quantified by phosphorimaging. The remaining MSS was
used to isolate DIGs as described for B, and proteins were
immunoprecipitated from gradient fractions 5-6 (detergent-insoluble
(I)) and 11-12 (detergent-soluble (S)). The
proportion of DIG-associated Pma1p and Sec22p was quantified relative
to the total amount present in the MSS + nucleotide.
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Fig. 4.
Oligomeric Pma1p is packaged into COPII
vesicles. A, microsomal membranes generated from either
wild-type (WT; RSY1578) or lcb1-100 cells at
24 °C were used for budding reactions performed with purified COPII
(+Lst1p complex) in the presence (+) or absence ( ) of nucleotide.
One-tenth of the total (T) and one-half MSS were solubilized
with Triton X-100 and analyzed by BN-PAGE and immunoblotting.
B, lst1-null cells (RSY1801) were pulse-labeled
for 3 min after treatment with myriocin (myr; 10 µg/ml) or
methanol only for 2 h and converted to permeabilized spheroplasts
for budding reactions. Duplicate reactions contained a constant amount
of Sar1p and Sec13p-Sec31p (20 µg/ml each), nucleotide, and
increasing amounts of both Sec24p and Lst1p complexes (0-20 µg/ml
combined). HA-Pma1p immunoprecipitated from the MSS was quantified by
phosphorimaging. C, shown are the results from BN-PAGE of
budding reactions from lst1 microsomal membranes in the
presence of Sar1p and Sec13p-Sec31p (20 µg/ml each) and either a
mixture of Lst1p and Sec24p complexes (10 µg/ml each) or Sec24p
complex alone (20 µg/ml). One-twentieth of the total (T)
and one-half MSS (+ or -nucleotide) were analyzed by immunoblotting
with HA-specific antibodies. D, cells treated as described
for B were used for budding reactions with constant levels
of Sar1p and Sec13p-Sec31p (20 µg/ml each) and low-to-intermediate
amounts of both Lst1p and Sec24p complexes or the Sec24p complex alone.
Budding of both oligomeric and monomeric HA-Pma1p was stimulated by
Lst1p, whereas budding in the absence of nucleotide (nuc)
was minimal.
cells treated with
myriocin and incubated with low-to-intermediate concentrations of
Sec24p and Lst1p complexes. Under these conditions, Pma1p was not
significantly packaged when only the Sec24p complex was added (Fig.
4D); however, addition of equivalent amounts of both Sec24p
and Lst1p complexes stimulated budding ~3-fold in both
myriocin-treated and control membranes. We conclude that the oligomeric
state of Pma1p does not enhance budding and that both the oligomeric
complex and the monomeric protein require Lst1p for efficient
incorporation into COPII vesicles.
Lst1p generates larger COPII vesicles independently of Pma1p
oligomerization
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a fellowship from the Human Frontiers Science Program.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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