* Section of Microbiology, University of California, Davis, California 95616; Department of Chemical and Biological Sciences,
Faculty of Science, Japan Women's University, Tokyo 112, Japan; and § Department of Cell Biology, National Institute for Basic
Biology, Okazaki 444, Japan
The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.
The yeast vacuole plays a key role in cellular protein
metabolism. During periods of nitrogen starvation,
amino acid supplies are maintained by protein degradation reactions that take place in this organelle (Klionsky et al., 1990 API is synthesized as a cytosolic precursor containing an
amino-terminal propeptide that mediates its vacuolar delivery (Segui-Real et al., 1995 Two lines of evidence suggest that API transport may be
a vesicular event. First, analysis of cytoplasm to vacuole
targeting mutants (cvt; Harding et al., 1995 The second indication that API transport is vesicular
comes from recent analysis of the biogenesis of this protein. These studies indicate that prAPI is transported as a
732-kD dodecamer (Kim et al., 1997 The degree to which the Cvt pathway is analogous to
bulk macroautophagy remains to be determined. There
are several fundamental differences between these two
pathways. API transport is a specific biosynthetic event
that is constitutive, occurring even under nutrient-rich
conditions (Klionsky et al., 1992 To provide direct evidence that API in fact uses a vesicular mechanism for vacuolar delivery, we have examined
the API transport process biochemically and morphologically. We have identified two yeast mutants that trap
prAPI inside a vesicular compartment and used one of
these mutant strains to purify an API transport intermediate. In addition, these data are supported by immuno-EM images demonstrating that API is concentrated in vesicles
during transport to the vacuole.
Strains and Media
Saccharomyces cerevisiae strains used in this study were: JSR18 Reagents
Gold-conjugated goat anti-rabbit IgG was from Biocell (Cardiff, UK). LR
white resin was from London Resin (Hampshire, UK). Optiprep was from
Accurate Chemical and Scientific Corp. (Westbury, NY); Oxalyticase
from Enzogenetics (Corvallis, OR); Expre35S35S was from Dupont-New
England Nuclear Research Products (Boston, MA); proteinase K and Pefabloc were from Boehringer Mannheim Biochemicals (Indianapolis, IN);
SYPRO orange was from BioRad Laboratories (Hercules, CA); and all
other reagents were from Sigma Chemical Co. (St. Louis, MO). Antiserum against peptides in the mature region of API (Klionsky et al., 1992 Pulse-Chase Analysis and Immunoprecipitation
For whole cell labeling experiments, yeast strains were grown to 1 OD600/ml
in SMD. Cells were harvested and labeled with 100 µCi/ml Expre35S35S in
SMD at a cell density of 100 OD600/ml for 5 min. Cold cysteine and methionine were added to 2 mM and 1 mM, respectively. The labeled cells
were chased at a cell density of 1 OD600/ml in SMD for the indicated
times. Where indicated, SD-N was used for chase reactions. Spheroplast
labeling was performed as described (Scott and Klionsky, 1995 Differential Lysis Fractionation
Fractionation experiments were performed using previously described
methods based on differential osmotic lysis (Harding et al., 1995 Subvacuolar Vesicle Isolation
Vacuoles were isolated from 700-800 OD600 U of spheroplasts prepared
from the cvt17 strain. Differential lysis was performed in PS200 containing
1 mM Pefabloc and 2 µg/ml pepstatin A, and the resulting P200 fraction
was resuspended in 3 ml of 12% Ficoll. This was overlaid with 3 ml of 10%
Ficoll, 3 ml of 4% Ficoll, and 1 ml of PS200. All Ficoll solutions were
made in PS200 with protease inhibitors. Vacuoles were isolated from the
4% Ficoll/PS200 interface after centrifugation at 100,000 g for 90 min at
8°C. The vacuoles were diluted with 2 vol of PS200 and collected by centrifugation at 60,000 g for 15 min. An aliquot of the vacuole fraction was
saved for analysis. The remaining vacuoles were resuspended in 200 µl
PS0 containing protease inhibitors and loaded on top of an Optiprep step
gradient consisting of 500 µl of 37% Optiprep in PS0 (density 1.12) overlaid with 1.3 ml of 19% Optiprep in PS0 (density 1.06). The resulting step
gradient was subjected to centrifugation at 160,000 g for 60 min at 12°C.
Four fractions were collected: the PS0/1.06 interface (vacuolar vesicles
[VV]), an equal aliquot of the 1.06 density region (low density [LD]), the
1.06/1.12 interface (subvacuolar vesicles [SV]), and the 1.12 density region
(high density [HD]).
Vacuole Isolation
Vacuoles were isolated from small scale preparations for the experiments
in Figs. 2 and 3. Spheroplasts (50 OD600 U) were lysed in PS200 buffer and
centrifuged at 5,000 g for 5 min to generate the P200 fraction. This fraction
was resuspended in 900 µl 10% Ficoll, and then overlaid with 1 ml 4% Ficoll and 200 µl PS200. The resulting step gradient was centrifuged at
166,000 g for 60 min at 8°C. The vacuole fraction (V) was collected from the 0/4 % Ficoll interface, the intermediate fraction (I) from the 4/10% Ficoll interface, and the pellet fraction (P) from the gradient pellet.
Protease treatment was with 50 µg/ml proteinase K for 15 min on ice.
Where indicated, Triton X-100 was added at a final concentration of
0.2%. Protease treatment results in the degradation of precursor API to
intermediate- and mature-sized bands; mature API is resistant to digestion by proteinase K (Oda et al., 1996 Immuno-EM
Samples were prepared for immuno-EM as described (Baba et al., 1994 API Maturation Is Severely Inhibited by
Low Temperatures
One way to distinguish between membrane translocation
through a proteinaceous channel and vesicular transport
reactions is by sensitivity to low temperature. For example, at 10°C translocation of CPY into the ER lumen occurs efficiently, while the packaging and transport of CPY
from the ER to the Golgi (a vesicular process) is blocked
(Baker and Schekman, 1989
Identification of Prevacuolar API-containing Vesicles
If API is transported to the vacuole by a vesicular intermediate, it may require some of the same components as the
vacuolar protein sorting (Vps) pathway for correct compartment identification, docking, or fusion. Similarly, if API-containing vesicles fuse at the endosome or late endosome
instead of at the vacuole, then proteins that are required
for late steps in the Vps pathway would also be required
for API targeting. Previously, we examined vps mutants
for their API phenotype. The vps mutants display differential effects on API processing; some vps mutants are essentially wild type for API transport, while others are
strongly blocked (Klionsky et al., 1992 vps18 To determine the step in the API transport pathway that
is affected by vps18, subcellular fractionation experiments
were performed. Spheroplasts were pulse labeled and lysed
in PS0, a buffer of low osmotic strength that lyses both the
plasma membrane and the vacuole membrane, while
smaller vesicles remain intact (see Fig. 4). After a 5-min
pulse at the nonpermissive temperature, the majority of
prAPI remained in the supernatant fraction and was sensitive to added protease, consistent with a cytosolic localization (Fig. 2 B). After 60 min of chase, a fraction of prAPI
was contained in the pellet fraction and was protease protected after lysis in PS0 buffer. Since this treatment lyses
both the plasma membrane and the vacuole, this result
suggests that prAPI is trapped in a prevacuolar vesicular
compartment.
To confirm that the population of prAPI contained
within the pellet fraction is not within the vacuole, further
fractionation experiments were performed. These experiments were carried out using a differential lysis technique
based on resuspension of spheroplasts in PS200 buffer.
Marker protein analysis shows that this treatment lyses the
plasma membrane, while maintaining the integrity of the
majority of vacuoles (Harding et al., 1996 A Targeting Mutant in the API Propeptide Accumulates
in a Nonvacuolar Compartment
To extend our analysis of the cytoplasmic intermediate
compartment, we examined the location of API with a mutation in the propeptide region. Mutations in the API proregion interfere with the membrane binding or subsequent
steps of the transport process. In particular, changing proline 22 to leucine (P22L) causes prAPI to accumulate in a
membrane-bound state that undergoes further transport
at a very minimal rate (Oda et al., 1996 Since P22L API is transport defective (Oda et al., 1996 Identification and Purification of Subvacuolar Vesicles
Containing API
Genetic and phenotypic analyses indicate that the majority
of the cvt, apg, and aut mutants are defective in both API
maturation and autophagy (Harding et al., 1996 When cvt17 cells were subjected to differential osmotic
lysis in PS200 buffer, the majority of prAPI and PrA was
maintained within the pellet fraction (Fig. 4 A), while cytosolic proteins such as PGK were released into the supernatant fraction (data not shown). The prAPI in these cells
was protease protected, indicating its localization to a
membrane-enclosed compartment (Fig. 4 A). Additional
fractionation experiments were performed to determine whether this population of API was localized in the vacuolar lumen or in a separate protease-protected compartment.
For these studies, cvt17 spheroplasts were resuspended
first in PS200 to lyse the plasma membrane while maintaining vacuolar integrity. After separating the cytosolic
fraction from the vacuole-containing pellet fraction by
centrifugation, the resulting pellet (P200) was subjected to a
second lysis step with a very low osmotic strength buffer
(PS0). After separation by centrifugation at 10,000 g, this
second lysis step resulted in the S0 and P0 fractions. While the majority of the vacuolar lumen marker PrA was recovered in the P200 fraction, it was released into the S0 fraction
upon resuspension in PS0, indicating vacuolar lysis. However, a considerable amount of prAPI is still maintained
within the pellet fraction, indicating that it is not free in
the vacuolar lumen (Fig. 4 B). The pelletable prAPI is released into a soluble fraction by treatment with Triton X-100, indicating that it is enclosed within a membrane
rather than an aggregate (data not shown). In addition,
prAPI is collected in the pellet fraction by the relatively
low force of centrifugation at 10,000 g, consistent with its
association with a large compartment such as an autophagic body. Microscopy studies indicate that autophagic bodies are ~ 300-900 nm in diam (Baba et al., 1994 The S0 and P0 fractions were subjected to protease treatment with proteinase K. The prAPI that was released into
the S0 fraction was digested to the mature size, while
prAPI in the P0 fraction was protease protected unless
membranes were solubilized by the addition of detergent
(Fig. 4 B), suggesting that the prAPI in the P0 fraction remains in a vesicular compartment. This result, together
with the fact that the prAPI is in a less osmotically sensitive compartment than the vacuolar lumen, suggests that it may instead be within a subvacuolar vesicle such as the autophagic bodies previously identified microscopically.
Purification of Subvacuolar Vesicles from cvt17 Cells
Since purified vacuoles contain little contamination from
other organelles, we used isolated vacuoles from cvt17
cells as the starting material for purification of prAPI-containing subvacuolar vesicles. Isolated vacuoles were permeabilized by resuspension in PS0 buffer to release API-containing vesicles. Optiprep step gradients were then
used to separate the released subvacuolar vesicles from remaining vacuolar membranes, which may reseal into vesicles after lysis and release of luminal contents. Appropriate densities for separation of these two vesicle populations in
our buffer system were determined empirically.
We found that vacuolar vesicles are unable to enter an
Optiprep solution with a density of 1.06, while some of the
prAPI-containing subvacuolar vesicles sediment through
the 1.06 density step and become concentrated at the 1.06/
1.12 interface (Fig. 5 A). The majority of vacuolar membranes in this gradient, marked by ALP, is in the VV fraction collected from the 0/1.06 density interface. This is in
contrast with prAPI, which is localized in both the VV
fraction and the SV fraction (the 0/1.06 and the 1.06/1.12 density interfaces). A portion of the VV fraction and the
SV fraction was subjected to protease treatment. Precursor API in both fractions was protease protected in the absence of detergent, indicating that it is still localized within
a membrane-bound compartment (Fig. 5 B). ALP contained in the VV fraction was digested to a slightly lower
molecular weight form both in the presence and absence
of detergent. As ALP is a type II transmembrane protein that contains a cytosolic domain (Klionsky and Emr, 1989
API-containing Vesicles Can Be Detected
by Immuno-EM
To provide additional evidence for a vesicular mode of
API transport, cells were examined using immuno-EM with
antibodies against API. Wild-type and cvt17 cells were grown
in YPD to late log phase, harvested, and rapidly frozen.
When wild-type cells were probed with preimmune serum,
or ape1
Characterization of prAPI-containing Vesicles
Additional analysis of the subvacuolar vesicles isolated
from cvt17 cells was performed to evaluate the degree of
purity attained by this fractionation procedure. After differential osmotic lysis and recovery on Optiprep step gradients, fractions were analyzed by immunoblot analysis
with antibodies against marker proteins for various yeast
organellar compartments. As in the experiment shown in
Fig. 5, the SV fraction was nearly devoid of PrA and ALP,
but still contained prAPI (Fig. 7 A). When these same
fractions were probed with antibodies raised against Kex2p,
a Golgi-resident protein (Redding et al., 1991
Recent evidence that the majority of mutants in the autophagy and Cvt pathways display the same phenotypes
suggested that API transport is vesicle mediated (Harding
et al., 1996 We performed fractionation analysis on various protein
targeting mutants to identify those that might accumulate
transport intermediates on the API targeting pathway. Examination of prAPI that accumulates in the vps18 mutant
revealed that a population of prAPI is trapped in a nonvacuolar vesicular compartment in this strain (Fig. 2). Although
the precise role of Vps18p is not known, it is involved at a
late step in vacuolar protein sorting, presumably after the
endosome (Preston et al., 1991 We predicted that, analogous to macroautophagy, API
is transported inside double-membraned vesicles (autophagosomes) that are formed in the cytosol. The autophagosomes would be subsequently targeted to the vacuole
where the outer membrane would fuse with the vacuolar
membrane, releasing the inner vesicle (autophagic body)
into the vacuolar lumen. This hypothesis is supported by subcellular fractionation experiments using cvt17 that demonstrate the presence of API-containing subvacuolar transport vesicles (Figs. 4 and 5). In addition, immuno-EM of
cvt17 confirmed the presence of API-containing subvacuolar vesicles (Fig. 6). Purification and characterization of
the complement of proteins contained in this compartment suggest that proteins unique from those of the vacuole make up these subvacuolar transport vesicles (Fig. 7).
Together, these data indicate that API is constitutively
packaged into cytosolic vesicles that are then transported
to the vacuole. These vesicles could either be targeted directly to the vacuole, or could first fuse with an endosomal
compartment. In the event that they fuse at the vacuole,
they may use common recognition and fusion components
such as a vacuolar t-SNARE and v-SNARE, as well as the
sec17/sec18 complex, to facilitate this reaction. If fusion instead occurs at the endosome, then all of the components necessary for late steps in vacuolar targeting or maturation
would be required for vacuolar delivery. This is similar to
the situation that has been proposed in mammalian cells,
where lysosomes develop through the fusion of various
different classes of endosomal compartments (Dunn, 1995 These results clearly demonstrate that API transport is a
vesicular process that has many similarities to the degradative macroautophagic pathway. Despite this evidence, the
relationship between the constitutive API delivery pathway and macroautophagy remains unclear. In addition,
many uncertainties remain regarding protein trafficking
by autophagic vesicles. These include such basic mechanistic questions as the membrane of origin of autophagic vesicles and what triggers the formation, movement, fusion, and, finally, breakdown of these transport vesicles. The availability of purified vesicular compartments and the use of API
as a marker protein will be valuable tools for these studies.
; Hilt and Wolf, 1992
). Accordingly, the
vacuole contains numerous hydrolytic enzymes, compartmentalized to prevent nonspecific cellular damage. Substrate proteins are delivered to the vacuole from the cytosol by autophagy (Takeshige et al., 1992
; Noda et al.,
1995
), and from the cell surface by endocytosis (Riezman,
1993
). Precise subcellular transport reactions are necessary to ensure both the faithful compartmentalization of
hydrolytic enzymes and the recognition and timely delivery of protein substrates for degradation. The majority of
vacuolar hydrolases are delivered to the organelle via the
secretory pathway (Conibear and Stevens, 1995
; Stack et al.,
1995
). Aminopeptidase I (API)1, however, does not use
the secretory system and is instead targeted by a nonclassical pathway from the cytoplasm to the vacuole (Klionsky et al., 1992
).
; Oda et al., 1996
). The proregion consists of two predicted
helices separated by a
proline residue. The first helix is amphipathic and directs
the binding of API to a target membrane (Oda et al.,
1996
). Upon arrival in the vacuolar lumen, the proregion
is cleaved in a proteinase B (PrB)-dependent reaction (Klionsky et al., 1992
). Thus, maturation of API is a convenient marker for correct vacuolar delivery.
) indicates that
there is substantial overlap between genes involved in API
transport and those required for autophagy (Harding et al.,
1996
; Scott et al., 1996
). In addition, phenotypic examination of cvt and autophagy mutants (apg, aut) revealed that
the majority of these mutants are both autophagy defective and accumulate API precursor (prAPI) (Harding et al., 1996
; Scott et al., 1996
). The apg mutants (Tsukada and
Ohsumi, 1993
) and, presumably, aut mutants (Thumm et al.,
1994
) are defective in macroautophagy, a process by which
cytosolic proteins and cytoplasmic organelles are engulfed
by double-membraned vesicles and delivered to the vacuole for degradation (Takeshige et al., 1992
; Baba et al.,
1994
).
). Since this complex
is much too large to penetrate a typical translocation channel, and large porelike structures cannot be present on an
energetically active membrane such as the vacuole, API
must be transported by a vesicular mechanism.
; Scott et al., 1996
). Bulk
autophagy is nonselective, degradative, and only detectable during starvation for nutrients such as nitrogen or
carbon (Takeshige et al., 1992
). Also, prAPI has a half-time for vacuolar delivery of 30-45 min (Klionsky et al.,
1992
), while the half-time for delivery of cytosolic proteins
by macroautophagy is at least 12 h when induced by nitrogen starvation (Scott et al., 1996
). One possible model that
accommodates these differences is that the Cvt pathway
uses essentially the same molecular mechanism as macroautophagy to deliver API to the vacuole. The transport
of API could be made specific, rapid, and constitutive if
API is concentrated into vesicles by a receptor-mediated
mechanism, and the constitutive level of autophagic vesicle formation is sufficient for delivery of API during exposure to various nutrient conditions.
Materials and Methods
1 MAT
vps18
1::TRP1 leu2-3,112 ura3-52 his3-
200 trp1
901 lys2-801 suc2-
9
with a plasmid bearing a temperature-sensitive allele of vps18, pJSR9
(Robinson et al., 1991
); SEY6210 MAT
leu2-3,112 ura3-52 his3-
200
trp1-
901 lys2-801 suc2-
9 GAL (Robinson et al., 1988
); SEY6211 MATa
leu2-3,112 ura3-52 his3-
200 trp1-
901 ade2-101 suc2-
9 GAL (Robinson
et al., 1988
); THY32 cvt17-1 derivative of SEY6211 (Harding et al., 1995
,
1996
); and DYY101 MAT
leu2,3-112 ura3-52 his3-
200 trp1-
901 ade2-101 suc2-
9 GAL ape1
::LEU2 with a single copy plasmid containing the
P22L API construct (Oda et al., 1996
). Unless otherwise indicated, cells
were grown in synthetic minimal medium (SMD; 0.067% yeast nitrogen
base, 2% glucose, and auxotrophic amino acids and vitamins as needed).
Synthetic minimal medium without amino acids or ammonium sulfate
containing 2% glucose (SD-N) was used for nitrogen starvation experiments, and YPD (containing 1% yeast extract, 2% peptone, and 2% glucose) was used for cell growth before immuno-EM.
),
API propeptide (Harding et al., 1995
), carboxypeptidase Y (CPY) and
proteinase A (PrA) (Klionsky et al., 1988
), and alkaline phosphatase (ALP)
(Scott et al., 1996
) were as described previously. The following antisera were generously supplied as indicated: phosphoglycerate kinase (PGK) (J. Thorner, Department of Biochemistry, University of California, Berkeley), Sec13p (C. Barlow, Dartmouth Medical School, Hanover, NH),
Kex2p (G. Payne, Department of Biological Chemistry, University of California, Los Angeles), and Sec61p (R. Schekman, Department of Molecular and Cellular Biology, University of California, Berkeley).
). Samples
were immunoprecipitated as described (Klionsky et al., 1992
).
; Oda et al.,
1996
). Spheroplasts were permeabilized in 200 mM sorbitol, 20 mM K-Pipes,
pH 6.8 (PS200), and soluble (S200) and organelle-containing (P200) fractions were collected by centrifugation at 5,000 g for 5 min. This treatment
consistently releases >95% of the cytosolic marker PGK into the supernatant while maintaining ~70% of the vacuole lumen marker PrA in the pellet fraction (Harding et al., 1995
, 1996
; Oda et al., 1996
). Where indicated,
a second osmotic lysis step was performed by resuspending the P200 fraction in 20 mM K-Pipes, pH 6.8 (PS0), and separating the S0 and P0 fractions by centrifugation at 10,000 g for 5 min.
Fig. 2.
Precursor API is trapped in a prevacuolar compartment
in vps18 cells. (A) Yeast strain JSR181 with plasmid pJSR9
(vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase
times. (B) vps18 spheroplasts were shifted to 38°C for 5 min,
pulse labeled for 5 min, and then chased for either 0 or 60 min. At
each time point the spheroplasts were collected by centrifugation
and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 µg/ml proteinase K, or
received proteinase K in the presence of 0.2% Triton X-100. The
supernatant (S0) and pellet (P0) fractions were collected after
centrifugation at 10,000 g for 5 min. (C) vps18 spheroplasts were
pulse labeled for 10 min, chased for 10 min at 26°C, and then
shifted to 38°C and chased for an additional 60 min. These cells
were then lysed in PS200 buffer, and the S200 and P200 fractions
were collected by centrifugation at 5,000 g for 5 min. The P200
fraction was resuspended in 10% Ficoll solution made in PS200,
and vacuoles were isolated by flotation through 4% Ficoll. V,
vacuole fraction; I, 4/10% Ficoll interface fraction; P, gradient
pellet. Proteins of interest were recovered by immunoprecipitation, followed by SDS-PAGE, and detected by a Molecular Dynamics STORM phosphorimager. The positions of precursor and
mature API and CPY are indicated.
[View Larger Version of this Image (41K GIF file)]
Fig. 3.
A mutant in the
API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A)
Spheroplasts from an ape1
strain containing a plasmid
bearing P22L API were subjected to differential lysis in
PS200 buffer. The Total (T)
fraction (after differential lysis) either received no treatment, was treated with 50 µg/
ml proteinase K, or received
proteinase K in the presence
of 0.2% Triton X-100. (B)
The supernatant (S200) and
pellet (P200) fractions were
collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll.
V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and
mPrA are indicated. (C) ape1
cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and
subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.
[View Larger Version of this Image (17K GIF file)]
). Immunoblotting was performed as
before (Harding et al., 1996
).
)
with modifications. Cells were sandwiched between an aluminum and a
copper disk, frozen in liquid propane, and transferred to 0.1% formaldehyde in absolute acetone at
80°C. After 1-2 d, they were transferred to
20°C, washed with absolute acetone, and then replaced stepwise with
cold absolute ethanol. LR white resin was applied and allowed to polymerize for 48 h with UV irradiation at
20°C. Ultrathin sections were collected onto Formvar-coated nickel grids, blocked with 2% BSA-PBS with
purified goat serum (1:50 dilution), and immunolabeled with affinity-purified API antibody by floating the grids on a 1:200 or 1:500 dilution of antiserum for 1 h at room temperature. The grids were then washed, incubated with 10 nm gold-conjugated goat anti-rabbit IgG, washed again,
and then fixed with 1% glutaraldehyde for 3 min. Sections were stained
for 7 min with 4% uranyl acetate and with 0.2% lead citrate for 30 s, and
then examined with an electron microscope (H-800; Hitachi Ltd., Tokyo,
Japan) at 125 kV.
Results
). We have previously shown that API maturation in vitro is sensitive to low temperature (Scott and Klionsky, 1995
). To examine the effect of
low temperature on the kinetics of API transport in cells,
pulse-chase experiments were performed at 10°, 14°, and
30°C. At 30°C API has a half-time for vacuolar delivery of
30-45 min (Klionsky et al., 1992
); the reaction is essentially complete after 2 h of chase (Fig. 1). However, at
10°C no mature API was detected, after incubation for 4 h
(Fig. 1). When CPY transport was also monitored in the same samples, the ER form of CPY (p1CPY) accumulated, indicating that membrane translocation into the ER
continued while vesicular reactions that carry cargo from
the ER to the Golgi were blocked (data not shown). When
the reactions were carried out at 14°C, a trace of mature
API was apparent at the 4-h chase point, indicating that
even at this intermediate temperature API delivery is severely inhibited. This requirement for relatively high temperatures is typical of vesicle-mediated transport but not
membrane translocation through a proteinaceous channel.
Fig. 1.
API delivery is blocked at low temperatures. SEY6210
cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and
30°C. API was recovered by immunoprecipitation followed by
SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and
mAPI are indicated.
[View Larger Version of this Image (40K GIF file)]
; Scott and Klionsky, 1995
). To reduce the possibility of secondary defects
and to allow a more direct assessment of the role of a vps
gene product in API import, we examined API delivery in
a vps18 temperature-conditional mutant.
cells containing a temperature-sensitive allele of
vps18 on a plasmid were subjected to pulse-chase experiments at the permissive and nonpermissive temperatures.
The vps18 strain was completely blocked for API delivery
at the nonpermissive temperature (Fig. 2 A). Previous
studies of Vps18p (Pep3p) suggest that it is involved in
transport between the endosome and the vacuole (Robinson et al., 1991
), and that it may be located on the vacuolar membrane surface (Preston et al., 1991
). The fact that API
transport is tightly blocked in this mutant suggests that
API-containing vesicles may either join vacuole-destined
proteins somewhere within the endosomal system, or may
use some of the same molecular components used by vesicles traveling from the endosome to the vacuole to fuse at
the vacuolar membrane.
Fig. 4.
Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment
in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to
differential lysis and protease treatment in PS200 buffer as in Fig
3. (B) Precursor API is not free in the vacuolar lumen in cvt17
mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant
(S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0
and P0 fractions were treated with proteinase K in the presence
and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of
interest were detected by Western blotting. The positions of
prAPI, mAPI, and mPrA are indicated.
[View Larger Version of this Image (18K GIF file)]
, 1995
). Cells
containing the vps18 mutation were pulse labeled for 10 min, and then chased for 10 min at the permissive temperature before shifting to the nonpermissive temperature for
60 min. This pulse-chase regime allowed for transport of
CPY to the vacuole to occur before the onset of the vps18
block. Since delivery of API is slower than CPY, maturation of API was still prevented. As in the PS0 buffer, a
population of prAPI was maintained in the pellet fraction
after lysis in PS200 buffer. Vacuoles were then isolated
from the P200 fraction by flotation through 4% Ficoll. After this treatment, mCPY was recovered in the vacuole
fraction, while trapped p2CPY was recovered in the gradient pellet. In the same samples, none of the trapped prAPI
was collected in the vacuole fraction (Fig. 2 C). These results indicate that the vps18 block causes the accumulation
of prAPI in a nonvacuolar compartment, and suggest that
API may be transported to the vacuole via a vesicular intermediate.
). This mutation is
predicted to disrupt a
turn separating two
helices that make up the API propeptide and results in a mutant protein with increased membrane affinity compared with the
wild-type protein (Oda et al., 1996
). Differential lysis fractionation experiments were performed in PS200 buffer using cells containing P22L API. P22L API was primarily
precursor sized, but it was recovered in the membrane pellet fraction where it was protease sensitive, indicating that,
although it is membrane bound, it is not compartmentalized (Fig. 3 A). Under these same conditions, wild-type
precursor is located exclusively in the supernatant fraction
(Harding et al., 1995
, 1996
). Vacuoles were then isolated
from the pellet containing P22L API. As was the case with
the trapped prAPI in the vps18 mutant strain, precursor
P22L API did not cofractionate with vacuolar markers
such as PrA, but was instead recovered from the gradient pellet (Fig. 3 B), suggesting that prAPI is first localized to a nonvacuolar membrane.
),
it is possible that prAPI that accumulates in this mutant
may not be on the correct transport pathway. To examine
this question, the severity of the P22L defect was followed
by pulse-chase analysis. P22L API-containing cells were
pulse labeled for 5 min, and then subjected to a nonradioactive chase reaction in either nitrogen-containing or nitrogen starvation medium. Nitrogen starvation increases
the capacity of the API transport machinery in wild-type
cells (Scott et al., 1996
). In nitrogen-containing medium,
the transport of P22L API is slow but not completely defective. However, when the chase was performed in SD-N,
the import efficiency was significantly improved (Fig. 3 C).
The rate of API delivery in this experiment is too rapid to
be explained by bulk autophagy alone. These results indicate that the deficiency of P22L API can be overcome in
conditions where the capacity for API transport is increased, suggesting that the defective API is maintained
on the import pathway.
; Scott et al.,
1996
). Our results with vps18 and P22L API suggested that
API enters a cytoplasmic vesicle. If API uses the same
machinery as that used by the autophagic pathway, the cytoplasmic intermediate compartment may be an autophagosome (Baba et al., 1994
, 1995
). Autophagosomes are double-membraned structures that fuse with the vacuole,
resulting in the release of a subvacuolar single-membraned
vesicle termed an autophagic body (Takeshige et al., 1992
;
Baba et al., 1994
). This vesicle is subsequently degraded in
a PrB-dependent manner. Accordingly, mutations in either PrA or PrB cause the accumulation of autophagic bodies inside the vacuole (Takeshige et al., 1992
), as well
as causing a processing defect in API (Klionsky et al., 1992
).
Lack of API maturation in pep4 or prb1 mutant strains
could be the result of a direct processing block and/or inaccessibility of precursor API that is contained within intact
subvacuolar vesicles. To investigate whether API can be
trapped within a subvacuolar vesicle, additional fractionation studies were performed using the cvt17 mutant. Like
PrA and PrB mutants, cvt17 also accumulates autophagic
bodies in the vacuole (Harding et al., 1996
) but has the additional advantage that it is not defective in PrA, so that this
protein can be used as a marker protein for the vacuolar lumen.
, 1995
),
much larger than secretory vesicles that exist in 50- and
100-nm classes (Novick et al., 1980
).
),
this is the expected result, indicating that the added protease is working efficiently.
Fig. 5.
Precursor API is contained within subvacuolar vesicles
in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were
lysed in PS0 buffer, loaded on top of an Optiprep step gradient,
and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV,
1.06/1.12 interface; HD, 1.12 region. (B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.
[View Larger Version of this Image (19K GIF file)]
cells were probed with API antiserum, no immunogold signal was detected (data not shown). Wild-type
cells probed with API antiserum contained dispersed gold
particles in the vacuole lumen, confirming that mature
API is a soluble luminal hydrolyase and indicating that
API itself does not appear to aggregate, despite its dodecameric structure (Fig. 6 A). When sections from cvt17 cells
were examined, both cytosolic and subvacuolar API-containing vesicles were evident (Fig 6, B and C). The cytosolic vesicles appeared near the surface of the vacuole.
These are most likely double-membraned vesicles that are
approaching the vacuole surface for docking and fusion. Subvacuolar vesicles were detected in cvt17 cells, but not
in wild-type cells, confirming that the accumulation of
these vesicles is the result of mutation in the CVT17 gene.
Fig. 6.
Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and
prepared for microscopy as described in Materials and Methods.
(A) Wild-type cells probed with antibody against mature API.
(B) A cvt17 section showing a cytosolic vesicle containing prAPI
probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with
antibody against mature API. N, nucleus; V, vacuole; (arrows)
API containing vesicles. Bars, 0.5 µm.
[View Larger Version of this Image (62K GIF file)]
), it was only
detected in the total fraction. Similarly, Sec13p, a component of COPII-coated secretory vesicles (Barlowe et al.,
1994
), was primarily detected in the total fraction. In addition, components of the coatomer complex were detected
in the total fraction, but not in the VV and SV fractions (data
not shown). Sec61p, an ER membrane protein (Stirling et al.,
1992
), was found in all of the recovered fractions. Sec61p is not enriched in the vacuole fraction when compared
with the total fraction, however, suggesting that it is contaminating this preparation, rather than specifically purified. The cytosolic marker protein PGK also contaminates
the vacuole and subvacuolar fractions to a certain degree,
but is not specifically enriched. These fractions were also
examined by staining with SYPRO orange. The protein
profiles of the vacuolar vesicle and subvacuolar vesicle fractions were clearly distinct. A number of proteins appeared specifically enriched in the subvacuolar vesicle
fraction (Fig. 7 B).
Fig. 7.
Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V),
vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are
shown. Proteins were detected by immunoblotting as indicated in
the figure. (B) SYPRO orange staining detected by fluorescent
scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are
indicated on the left. Polypeptides enriched in the subvacuolar
vesicles are indicated on the right.
[View Larger Version of this Image (44K GIF file)]
Discussion
; Scott et al., 1996
). Additionally, studies of the
oligomeric state of API during transit indicate that this
protein is assembled into a dodecamer in the cytosol before transport (Kim et al., 1997
). These results, coupled with
the fact that API transport is extremely sensitive to low
temperatures (Fig. 1) and requires a GTP-binding protein
(Scott and Klionsky, 1995
), indicated that the Cvt pathway may be vesicular. In this report we demonstrate biochemically and morphologically that prAPI is transported by a
vesicle-mediated process.
; Robinson et al., 1991
).
This result, together with the fact that the P22L API mutant accumulates on a nonvacuolar membrane (Fig. 3), indicates that prAPI first binds and then becomes encapsulated in a vesicle before delivery to the vacuole. These
results are supported by immunoelectron microscope
analysis, which indicates the presence of API-containing
vesicles in the cytosol (Fig. 6 B).
).
Possibly the yeast endosomal system operates similarly: autophagic vesicles and secretory vesicles carrying vacuole-destined cargo could both fuse and deliver their contents to the endosome. Through continued fusion reactions,
this compartment could develop into an enzymatically mature vacuole. Further studies will provide information on
the mechanism of API import and in particular the stage
at which it integrates with vacuolar delivery through the secretory pathway and endosomal systems.
Received for publication 23 December 1996 and in revised form 23 April 1997.
1. Abbreviations used in this paper: ALP, alkaline phosphatase; API, aminopeptidase I; prAPI, API precursor; CPY, carboxypeptidase Y; HD, high density; LD, low density; PGK, phosphoglycerate kinase; PrA, proteinase A; PrB, proteinase B; SV, subvacuolar vesicle; VV, vacuolar vesicle; V, vacuole fraction; I, intermediate fraction; P, pellet fraction; Vps, vacuolar protein sorting.We thank Dr. Masako Osumi, Japan Women's University for the use of EM facilities, John Kim for helpful discussion and critical reading of the manuscript, and Drs. C. Barlow, G. Payne, R. Schekman, and J. Thorner for providing antisera.
This work was supported by Public Health Service grant GM53396 from the National Institutes of Health to D.J. Klionsky.
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