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INTRODUCTION |
Most vacuolar hydrolases are transported to the vacuole through
the endoplasmic reticulum and the Golgi apparatus. There are two
vacuolar enzymes known, which do not follow this pathway and are
transported from the cytoplasm to the vacuole independent of the
secretory pathway: aminopeptidase 1 (Ape1p)1 and
-mannosidase
(1, 2). The primary structure of N-terminal presequence of the Ape1p
precursor protein (pApe1p) indicated that the protein might not enter
the endoplasmic reticulum (3). Biochemical as well as genetic analysis
of pApe1p transport by Klionsky and colleagues (2) revealed that pApe1p
is transported to the vacuole independently of the Golgi or the
secretory plasma membrane route. A genetic screen based on the
detection of accumulated Ape1p precursor peptide by Western blot lead
to the isolation of Cvt mutants, defective in cytoplasm to
vacuole transport of pApe1p (4). These mutants
accumulate pApe1p in small double-membrane vesicles, Cvt vesicles,
similar to autophagocytic vesicles. Other genetic screens for cytoplasm
to vacuole protein transport are designed to study the degradative
protein transport pathways essential for yeast growth under nutrient
starvation conditions. Mutants unable to survive nutrient starvation
were analyzed for defects in autophagocytosis and accumulation of
double-membrane vesicles (apg mutants) (5). A cytoplasmic
protein degraded by this pathway is fatty acid synthetase, and
transport mutants for this protein were isolated (aut
mutants) (6). The cytosolic fructose-1,6-bisphosphatase can be degraded
in vacuoles, and mutants of this pathway (vid) were also
isolated (7). The vacuolar biogenesis-defective Cvt mutants and the
mutants of the autophagic pathways overlap genetically, demonstrating
that these pathways share components (8, 9).
The pApe1p monomer assembles with a half-time of 2 min into a
homododecameric complex in the cytoplasm, but the half-time for
vacuolar delivery is 45 min (10). pApe1p has been detected in
cytoplasmic double-membrane vesicles by immunoelectron microscopy, and
pApe1p is seen in the vacuole of protease-deficient cells within
single-membrane vesicles, indicating that the cytoplasmic pApe1p
dodecamer is enwrapped by the membrane of an autophagosome, which is
transported to and fuses with the vacuolar membrane (11-13). The
61-kDa cytoplasmic pApe1p is proteolytically matured to a 55-kDa
intermediate form and to the 50-kDa vacuolar mApe1p form by the
sequential action of the two vacuolar endopeptidases PrA and PrB (14).
The 45-amino acid precursor sequence forms a helix-turn-helix structure. Mutations that disturb formation of the first helix also
abolish pApe1p transport (15, 16). Ape1p is only found as
homododecameric complexes, and the dodecameric state is required for
its enzymatic activity (17). The HSC70 proteins Ssa1p and Ssa2p are
required for pApe1p transport, however, they do not control
dodecamerization of pApe1p but are required for vacuolar transport of
pApe1p by Cvt vesicles (18).
We developed a new genetic screen for analyzing the biogenesis pathway
of aminopeptidase 1 based on the enzymatic activity of the dodecameric
complex. We isolated two new mutants deficient in aminopeptidase 1 transport. Both display phenotypes, which have not been described for
Cvt or Apg and Aut mutants. The mutants were termed via for
vacuolar import and autophagocytosis. Mutants are defective in
dodecamer formation and pApe1p accumulates in Cvt vesicles, indicating
that complex assembly and transport vesicle formation are linked
processes. Dodecameric pApe1p is already proteolytically active, and,
therefore, packaging of pApe1p into Cvt vesicles is a pathway by which
the cell can control the potentially harmful proteolytic activity by
controlled assembly and compartmentalization.
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MATERIALS AND METHODS |
Strains, Media, and Plasmids--
SEY6211: MATa,
ura3-52, his3
1,
leu2-3,-112, trp1-289,
suc2-D9, ade (S. Emr); II-17: MAT
, ura3-52, his3
1,
leu2-3,-112, trp1-289,
lap1, lap2, lap3, lap4
(D. H. Wolf). These were mated and spores were isolated to
generate the strains PSY of a and
mating types deficient
in all four lap genes. These were transformed with the APE1
gene to generate the strains C6C MAT
and C6A
MATa.
Media were used as follows. YPD: 1% bacto-yeast extract, 2%
bacto-peptone, 2% glucose or 3% ethanol for YPE. MV-D: 0.67% yeast nitrogen base w/o amino acids, 2% glucose. MV-N: 0.17% yeast nitrogen base w/o amino acids, 2% glucose. All amino acids were added to the MV
media except the auxotrophic markers required. 2% agar was added for
plates. Media ingredients were purchased from Life Technologies, Inc.
Cell growth was determined by determining the optical density at 600 nm
and the glucose concentration.
The APE1 genomic locus, including the promotor region, was
isolated from a genomic DNA library (gift of H. D. Schmidt) by hybridization of the library with a polymerase chain reaction-amplified APE1 gene fragment. The locus was subcloned as a 5-kb
SalI fragment into the pRS centromere plasmids 313, 314, and
316. Mutations of the PEP4 gene were done with the pTS15
construct (19).
Mutagenesis and Colony Screening--
EMS mutagenesis was done
with stationary cultures of C6C in YPD as described. Cells were
resuspended in 0.1 M NaPi, pH 7.4, 100 µl EMS
(Merck) was added to 3-ml cells, and suspension was incubated over 30 min at 30 °C. Cells were washed three times with water, plated onto
YPD plates, and incubated for 3 days at 30 °C. Replicas were made on
MV-D/-Trp and incubated 4-7 days at 30 °C, and colonies were
transferred onto filters. These were placed with the colony side
up on YPD plates and incubated over night at 30 °C. For
colony activity assays clones were lysed in chloroform, air dried, and
covered with 0.7% agarose, 50 mM Tris/Cl, pH 7.5, 3 mM EDTA, 10 mM leucine-
-naphthylamide.
Activity was tested at 365 nm after incubation over 10 and 30 min at
25 °C. Fluorescence was recorded by a CCD chemiluminescence camera
system (Raytest, Straubenhardt, Germany), and CPY and CPS microtiter assays of mutant clones were done as described previously (20). Clones
deficient in Ape1p activity were transformed with a APE1-His plasmid after loss of the APE1-Trp plasmid present during
the mutagenesis and tested again for Ape1p activity. Mutants or spores were mated with C6C or C6A for tetrad analysis.
Biochemical Protocols and Cell Fractionation--
Ape1p activity
assays were done as described using leucine-
-naphthylamide (overlay
assay) and leucine-p-nitroaniline (spectrofluorometric assay) (Bachem) as substrates (21). Protein extracts were prepared after spheroplasting the cells: cells were incubated over 20 min at
room temperature in 0.1 M Tris/SO4, pH 9.4, 10 mM dithiothreitol to 1 ml/10
A600. Cells were harvested, resuspended
in 1.2 M sorbitol, 50 mM Tris/Cl, pH 7.5, 10 mM EDTA to 2 ml/10 A600. Lyticase
(Roche Molecular Biochemicals) was added to 0.125 µg/1
A600 and incubated at 30 °C over 30 min.
Spheroplasts were harvested, resuspended in the appropriate buffer, and
broken by vortexing the cells with 200-µl glass beads three times for
2 min with intermittent cooling on ice or lysed by osmotic lysis using
deionized water (1:1) or DEAE-dextran (90 µg/100
A600) (Sigma). In all experiments a protease inhibitor mix was used: 100 µM E-64, 20 µM leupeptin, 20 µM pepstatin, and 2 mM phenylmethylsulfonyl fluoride (BIOMOL). Protein
concentration was estimated using the Bio-Rad protein assay reagent.
Cell fractionation was essentially done as described by Scott et
al. (13) with the following modifications. The equivalent of 300 A600 of cells were used per gradient. The
spheroplasting was done with Zymolyase 20T (Seikagaku Corp., Japan) at
a concentration of 0.2 mg/50 A600 of cells. The
spheroplasts were lysed with DEAE-dextran at a final concentration of
60 mg/100 A600 of cells and were pelleted at
500 × g for 5 min. The pellet was resuspended in 4 ml
of 10% Ficoll. Unlysed spheroplasts were pelleted at 500 × g for 3 min, and the supernatant was transferred to a
Beckmann SW-40 centrifuge tube. The resuspension was overlaid with 5 ml
of 4% Ficoll and 2 ml of 200 mM sorbitol, 10 mM potassium PIPES, pH 6.8. The gradient was centrifuged at
30,000 rpm for 1 h and 30 min at 4 °C in the Beckman SW-40
rotor. The 0% Ficoll fraction, the 0/4% Ficoll interphase, and
the pellet fraction were collected, trichloroacetic acid-precipitated, resuspended in Laemmli buffer, and separated by a 10% SDS-PAGE.
A rabbit anti-Ape1p antisera was generated against amino acids 191-210
of Ape1p after coupling the peptide to hemocyanin. The pro-GFP
expression construct was a gift of I. Sandoval and M. Martinez, and
pro-GFP was detected with a mouse monoclonal anti-GFP antibody (Santa
Cruz Biotechnology). CPY and Hex antisera were a gift of H. D. Schmidt. Protein antibody complexes were made visible by decoration
with horseradish peroxidase-coupled secondary antibody (Dianova) and
chemiluminescence (Pierce Super-Signal). Exposed x-ray films were
scanned and quantified using Wincam software.
Protease Protection and Complex Assembly--
Spheroplasts were
prepared and lysed with DEAE-dextran as described above, but no
protease inhibitors were included. The lysed material was centrifuged
at 5000 × g over 5 min and lysis was analyzed by
Western blot analysis of pellet and supernatant for CPY (vacuolar) and
hexokinase. The lysed material was incubated at 4 °C over 30 min
without adding protease, in the presence of trypsin or proteinase K
(each 50 µg/ml) and in the presence of either proteinase and 0.2%
Triton X-100. Incubation was terminated by adding 1 volume of 20%
trichloroacetic acid/80% acetone. Processing of pApe1p was analyzed
after SDS-PAGE by Western blotting.
Glycerol gradients were done as described previously (10). A
step-gradient was formed by equal volumes of 50, 40, 30, and 20%
glycerol in 20 mM potassium PIPES, pH 6.8. 1.5 mg/400 µl
of crude cell extract proteins were loaded and centrifuged in a TLS-55 rotor at 55,000 rpm over 4 h at 15 °C in a Beckman table-top
ultracentrifuge. Gradient was collected in 10 fractions, and proteins
were trichloroacetic acid-precipitated and separated on a 10% SDS-PAGE
and analyzed by Western blot analysis. Ovalbumin (45 kDa), bovine serum
albumin (65 kDa), and thyroglobulin (669 kDa) were used to follow
separation on the gradient.
Vacuole Staining--
Vacuoles of cells were stained of mid-log
cultures in YPD. Cells were harvested and resuspended in 50 mM sodium citrate, pH 4, 1 µl of 10 mM
CL2CFDA (Molecular Probes) in Me2SO was added, and the cells were incubated at 30 °C over 10 min. Cells were harvested and resuspended in 0.1 M NaPi, pH
7.5, 2% glucose and visualized using a Zeiss Axiovert 100 microscope equipped with a Plan-Neofluar 63x/1,25 and
differential interference contrast.
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RESULTS |
Isolation of Aminopeptidase 1 Transport Mutants--
Ape1p is
synthesized as a cytosolic precursor protein, pApe1p, which assembles
into a homododecamer and is proteolytically matured after it reaches
the vacuolar lumen. Only this dodecameric complex is enzymatically
active (17). We designed a screen for mutants deficient in the
biogenesis pathway of Ape1p based on the enzymatic activity. The
sensitivity of the detection method allowed to screen under conditions
where autophagy is down-regulated. Cells were grown on filter paper
soaked with rich medium (YPD), lysed with chloroform, and filters were
covered with buffered 0.7% agarose containing the Ape1p substrate
leucine-
-naphthylamide. Enzymatic activity was detected by
the
-naphthylamide fluorescence. Yeast contains four
leucineaminopeptidases (APE1-APE4). To assay Ape1p activity, a plasmid carrying the APE1 gene under the
control of its endogenous promoter was transformed in a yeast strain
lacking these four leucineaminopeptidase activities (21). When this strain was transformed with one or two single-copy APE1
plasmids, the difference in the copy numbers was clearly detectable by
the fluorescence in the overlay assay for enzymatic activity. The overlay assay allowed us to visually detect differences of 20% of wt
Ape1p activity (Fig. 1B). This
observation was verified by determination of specific Ape1p enzymatic
activities in cell extracts. Isogenic strains of opposite mating types
deficient in the four leucineaminopeptidases were generated by mating
II-17 with SEY6211. Spores were tested for leucineaminopeptidase
activities using the overlay assay and recorded with a CCD
chemiluminescence camera system. Leucineaminopeptidase-deficient
strains do not show any phenotype related to vacuolar function as for
example growth under nitrogen limitation and sporulation.

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Fig. 1.
A, flow chart for the isolation of
via mutants (see text for details). B, Ape1p
"overlay" activity assay used for the isolation of Ape1p transport
mutants. The image was recorded at 365 nm using a CCD chemiluminescence
camera. Strains with Ape1p enzymatic activity emit fluorescent light,
and colony streaks become visible. Strains shown: OM is the
original via14 mutant isolated from the screen.
VIA14b spore is positive and via14a negative for
Ape1p activity. C6C is the wt strain. Diploids for
complementation analysis are the via14a/C6C wt cross and the
cross of via14a/via10b, a via10 spore
with reduced Ape1p enzymatic activity.
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The strain C6C carrying the APE1 gene on a single-copy
plasmid with tryptophan as auxotrophic marker was mutagenized by
EMS treatment (Fig. 1A). Cells were grown on rich media
(YPD) plates for 2-4 days at 30 °C. Replicas were made on synthetic
media (MV-D) containing all supplements except tryptophan to select for
the ectopic APE1 gene. From these plates replicas were made
on chromatography papers, which were placed on rich media plates (YPD)
to avoid up-regulation of nonspecific autophagocytosis. After
incubation of the filters overnight at 30 °C, colonies were tested
for Ape1p activity. Clones showing no and severely reduced Ape1p
activities were tested for the activities of the vacuolar
carboxypeptidases Y and S, which reach the vacuole by the secretory
pathway and which are proteolytically matured by the same vacuolar
endopeptidases as is pApe1p. This allowed us to identify and exclude
mutants defective in vacuole biogenesis due to mutations in the
secretory pathway. Clones were transformed with a APE1 gene
on a single-copy plasmid using histidine as an auxotrophic marker after
they had lost the tryptophan-plasmid to identify mutants of the
APE1 gene (Table I). We did
not isolate multiple alleles of the mutated genes, indicating that the
screen is not saturating and that mutations in this pathway are
synergistically lethal with mutations in other genes. We obtained 14 mutants, which were named via mutants for vacuolar import and
autophagocytosis mutants. Three rounds of tetrad analysis
of the mutants were performed using the isogenic wt strains to isolate
spores whose phenotype was caused by a single mutation. We describe the
analysis of two complementation groups, via10 and
via14 (Fig. 1B). Diploids of the via10
and via14 have 80% of aminopeptidase 1 activity. This could
be due to a gene dosage effect and indicates related functions of the
gene products.
via10 and via14 Mutants Accumulate pApe1p--
Mutants were
isolated based on the absence of Ape1p enzymatic activity. After tetrad
analysis, via14 cells did not show enzymatic activity,
whereas via10 cells had 20% of the wt activity. Crude cell
extracts were prepared from the strains in the presence of protease
inhibitors to analyze pApe1p processing. Proteins were separated by
SDS-PAGE and blotted onto nitrocellulose membranes, and Ape1p was
detected using a rabbit anti-Ape1p antiserum (Fig. 2). Cells were grown at 30 °C in
nutrient-rich medium (YPD) where autophagy is not active during
vegetative growth. wt yeast show 10-30% pApe1p and 70-90% mApe1p
under logarithmic growth conditions between A600 = 0.8-1.2 (YPD), whereas only 2% pApe1p can be detected in stationary
cultures with A600 > 2. via10
accumulates 60% of Ape1p as pApe1p, whereas via14
accumulates 90% as pApe1p under logarithmic growth conditions in
nutrient rich medium (YPD). Under stationary growth conditions in YPD
medium, pApe1p levels are reduced in via10 and in
via14 and mApe1p is detected even in via14, indicating that up-regulation of autophagy by glucose starvation in
via10 and via14 cells restores pApe1p transport.
Expression of pApe1p is induced by the starvation conditions, as is the
case for all vacuolar enzymes and proteins related to vacuolar function (26), but vacuolar transport is not comparably induced, so that pApe1p
accumulates. The ratio of pApe1p to mApe1p does change, and the
majority of Ape1p is present as mApe1p in stationary cultures. Cells
were grown in nitrogen starvation medium (MV-N) where autophagy is
up-regulated, and we tested for pApe1p (Fig. 2). In nitrogen starvation
medium pApe1p transport was enhanced in both via mutants, leading to mApe1p levels in via10 comparable to wild-type
and in stationary via14 cells comparable to mApe1p in
logarithmically growing wt cells. Note that we loaded 10 µg of wt
cell extract and 20 µg of mutant cell extracts on the gels used for
Fig. 2, as can be seen on the CPY loading control. This allowed the
quantitative comparison of the Ape1p signal intensities despite the
differences in expression levels between wt and via mutant
cells.

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Fig. 2.
Analysis of Ape1p transport.
Steady-state levels of pApe1p and mApe1p are detected by Western blot
analysis of protein extracts prepared from logarithmically
(L) and stationary (S) cultures grown in rich
media (YPD) and nitrogen starvation medium
(MV-N). Mutant strains express lower amounts of Ape1p. To
allow comparison of Ape1p signal intensities, different amounts of cell
extract proteins were loaded per lane: 10 µg of wt and 20 µg of
mutant cell protein extracts. The vacuolar CPY served as internal
control. Protein antibody complexes were detected by chemiluminescence
and x-ray film exposure.
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Figs. 2 and 4 demonstrate the reduced expression levels of Ape1p in the
via mutants. This could be caused by enhanced proteolytic turnover within the vacuole due to misfolding or disturbed complex assembly. Mutation of the vacuolar endopeptidase PrA (PEP4)
prevents degradation of the vacuolar autophagic bodies leading to the
accumulation of pApe1p within the vacuole (22). We mutated the PrA gene
PEP4 and tested for steady-state concentrations of Ape1p in
YPD. We found twice as much Ape1p in wt as well as mutant extracts, so the difference between wt and mutant cells remained (not shown). Therefore, vacuolar turnover did not contribute to the lowered Ape1p
levels in the mutant cells and degradation by cytoplasmic proteolytic
systems should account for the reduced expression levels (Fig.
3). We also tested for APE1
mRNA and could not detect differences between mutants and wt cells
(Ref. 28 and data not shown). In addition, expression of Ape1p is
induced in the mutant cells by starvation conditions as the vacuolar
CPY, indicating normal regulation of protein expression (Fig. 2).
Induction of pApe1p transport by autophagy in nitrogen starvation
medium protects pApe1p from degradation in the cytoplasm (Fig.
2).

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Fig. 3.
A, subfractionation of pApe1p and mApe1p
by 5000 × g centrifugation in a pellet (P)
and supernatant (S) fractions. CPY and hexokinase
(HK) were used as controls. Incubations for protease
protection were performed in the absence ( ) or presence of Triton
X-100 as detergent (Det.) and in the absence ( ) or
presence of trypsin or proteinase K. B, processing of pApe1p
by endogenous proteases in the absence ( ) or presence (+) of Triton
X-100 in strains with active proteinase A (PEP4) and with a
deleted proteinase A gene ( pep4).
p, pApe1p; i, intermediate form; m,
mApe1p.
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pApe1p Accumulates in Cvt Vesicles--
We analyzed whether the
pApe1p in the via mutants accumulates in vesicles by testing
the processing of the precursor protein through added trypsin or
proteinase K in the presence or absence of detergent (Fig.
3A). Logarithmically growing cells from rich medium (YPD)
were converted into spheroplasts and lysed with DEAE-dextran. Lysis of
spheroplasts was controlled by centrifugation of the lysate at
5000 × g and Western blot analysis of pellet and
supernatant fractions for the vacuolar carboxypeptidase Y (CPY) and
cytoplasmic hexokinase (HK). mApe1p and the majority of pApe1p was
found in the pellet fraction of wt cells, which is in agreement with
previous results (13). The same distribution of Ape1p proteins was
found in the via10 mutant. In the via14 mutant
the entire pApe1p was found in the supernatant after cell lysis (Fig.
3A).
The lysed cells were incubated for 30 min at 4 °C without proteinase
added, with added trypsin or proteinase K and with trypsin or
proteinase K in the presence of Triton X-100. In wt cells and in the
two via mutants, the same amount of pApe1p was
protease-protected in the absence of detergent (Fig. 3A).
Trypsin is not able to process pApe1p from wt cells in the presence of
detergent. pApe1p accumulating in via10 and via14
cells is readily processed by trypsin. pApe1p in wt cells was processed
by proteinase K in the presence of detergent. This indicated that
pApe1p accumulates in a non-native conformation in via10 and
via14 mutants.
We also tested whether endogenous proteases are able to process pApe1p
under the in vitro conditions of the protease protection assay by just adding detergent and no additional protease. Strains with
a deleted PrA gene (
pep4) served as control.
pApe1p processing of the wt strain was not detectable (Fig.
3B). pApe1p accumulated in via10 and
via14 was processed; however, this was not dependent on PrA
activity, as expected from the Ape1p levels detected in nitrogen
starvation medium and in
pep4 stains. This
demonstrates that the degradation of Ape1p in the via
mutants is caused by nonvacuolar proteases.
pApe1p in via14 did not cofractionate with the vacuolar CPY
and, therefore, accumulates in prevacuolar vesicles. To analyze whether
pApe1p in the via10 mutant accumulates in prevacuolar vesicles, organelles were fractionated on a ficoll gradient and pApe1p
and mApe1p fractionation was analyzed as previously described. In wt
cells vacuoles and mApe1p are found at the 0-4% Ficoll interphase, whereas Cvt vesicles and pApe1p are found in the pellet fraction (13).
pApe1p of via10 migrated into the pellet of the ficoll gradient while mApe1p migrated to the 0-4% Ficoll interphase as in wt
cells (Fig. 4A).

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Fig. 4.
pApe1p accumulates in prevacuolar vesicles in
via10 and via14 cells.
A, pApe1p localization after fractionation of wt
andvia10 organelles on a Ficoll gradient. L,
load; Vac, vacuolar 0/4% Ficoll; P, pellet;
p2CPY is the Golgi precursor form and mCPY is the
mature vacuolar CPY. B, pApe1p localization in
via14 cells after fractionation of cell lysates by 5000 × g and 100,000 × g centrifugation on a
10% Ficoll cushion. P, pellet; S, supernatant;
F, Ficoll. C, protection from proteinase K
digest of load and the fractions of the 100,000 × g centrifugation.
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In the via14 mutant pApe1p accumulates in prevacuolar
vesicles as in wt cells and via10, but they have a lower
density than wt Cvt vesicles and are found in a 5000 × g supernatant fraction (Fig. 3A). We tried to
pellet the via14 vesicles at 100,000 × g,
but pApe1p was not protease-protected anymore after the centrifugation, indicating that these vesicles are fragile (not shown). Therefore, we
spun the vesicles onto a 10% Ficoll cushion at 100,000 × g, 4 °C, and 30 min (Fig. 4B). 80% of pApe1p
was found at the 10% Ficoll fraction, 20% were in the supernatant,
and no pApe1p was found in the pellet. pApe1p in the supernatant was
not protease-protected, but pApe1p in the Ficoll fraction was
vesicular, although 50% of the fraction was degraded in the absence of
detergent (Fig. 4C). via14 vesicles appear to be
extremely fragile (compare Figs. 3A and 4B),
indicating that they lack essential components or that their assembly
is disturbed.
Deficient Cvt Vesicle Formation--
Enhanced cytoplasmic
degradation of pApe1p and vesicular accumulation of pApe1p could be
caused by a deficiency in Cvt vesicle formation. We expressed the
aminopeptidase 1 propeptide fused to GFP under the control of the
APE1 promoter from a single-copy plasmid in the cell lines.
The aminopeptidase 1 propeptide sequence targets the fusion protein to
the vacuole, but it is rapidly degraded in the cytoplasm and the
vacuole unless it is protected by the Cvt vesicle membrane (23). The
protein does not oligomerize, and cell lines expressing pro-GFP did not
express wt aminopeptidase 1 to exclude competitive inhibition of
transport. If Cvt vesicle formation is impaired in the mutants, pro-GFP
would be degraded, if Cvt vesicle formation is normal but transport is
impaired, more pro-GFP should be detectable in the via
mutants. Cell lines were grown in rich medium (YPD) to logarithmic
(A600 = 0.8) and stationary
(A600 > 2) phases, and pro-GFP was detected by
anti-GFP Western blot analysis. In all cell lines pro-GFP was detected migrating at 38 kDa as well as degradation products, but less protein
was detected in the via mutants (Fig.
5). This demonstrates that Cvt vesicle
formation is impaired in the via mutants. The pro-GFP
expression levels were insufficient to visualize vesicles by
fluorescence microscopy.

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Fig. 5.
Transport of pro-GFP in nutrient-rich media
(YPD) of control and via10 and
via14 cells. The upper band is the
38-kDa fusion protein, and the lower bands are degradation
products of pro-GFP. L indicates logarithmic and
S stationary growth phases.
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pApe1p Accumulates in a Non-native Conformation--
The in
vitro and the indicated in vivo protease sensitivity of
the pApe1p accumulating in the via10 and via14
mutant indicated that the protein is not in a native conformation.
pApe1p monomers assemble into a dodecameric pApe1p complex in the
cytoplasm with a t1/2 of 2 min, and enzymatic
activity of mApe1p requires dodecamerization (10, 17). We analyzed pApe1p complex assembly by separating Ape1p oligomers on a glycerol gradient as previously described (10). Crude protein extracts were
prepared from cells grown in YPD to A600 = 0.8-1.2 by breaking spheroplasts with glass beads in the presence of
protease inhibitors. 10 soluble fractions of the glycerol gradient were
collected, proteins were separated by SDS-PAGE, and Ape1p was detected
by Western blot analysis (Fig. 6).
Western blot signals on x-ray films were scanned, and signal
intensities were quantified (Fig. 5, Wincam 2.2 software). Three
experiments were performed with each genotype. In wt cells 90% of
Ape1p accumulated as dodecamers of pApe1p and mApe1p in fractions 5, 6, and 7 (Fig. 6A), and 10% of each pApe1p and mApe1p were
found in larger complexes in fraction 10 of the gradient. Also 90% of
the vacuolar mApe1p in the via10 and via14
mutants form stable dodecameric complexes. However, pApe1p in the
via mutants does not form stable dodecamers. 10% of total
pApe1p are found in fractions 3 and 4, and 20% of pApe1p are found in
fraction 10, demonstrating defects in complex assembly and formation of
high molecular weight aggregates (Fig. 6B). In the
via14 mutant 60% of total pApe1p was found in fraction 2 and fractions 9 and 10 of the gradient (Fig. 6C). The
additional bands (5% of total pApe1p) appearing in fraction 2 of the
gradient from via14 cells were not included in the
quantification. They probably represent degradation products of pApe1p
despite the fact that cell extracts were prepared in the presence of
protease inhibitors. Concentration of proteases in the upper fractions
of the gradient could lead to sufficient residual hydrolytic
activity.

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Fig. 6.
pApe1p and mApe1p oligomers separated on a
glycerol gradient. Gradient was collected in 10 fractions, which
were analyzed by Western blotting and quantified by scanning and
density determination of the x-ray films. A, quantification
and Western blot of the wt strain C6C; B, of the mutant
via10; C, of the mutant via14.
Upper part, for quantification the signals of pApe1p and
mApe1p were summarized separately to better demonstrate differences in
oligomerization. Numbers give the percentage of total pApe1p
(closed circles) or total mApe1p (open circles)
in the fractions. Numbers in kDa mark the position of the
markers ovalbumin (45 kDa), bovine serum albumin (65 kDa), and
thyroglobulin (669 kDa) on the gradient. Lower part, Western
blots of the gradient fractions.
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We observed increased pApe1p transport after induction of autophagy. To
test whether this transport also restores enzymatic activity and thus
dodecamerization of Ape1p, cell lines were grown on YPD and nitrogen
starvation medium to logarithmic and stationary phases, and colony
overlay assays for Ape1p enzymatic activity were performed (Fig.
7). Although Ape1p enzymatic activity in wt cells increased with incubation time in both media and was higher in
nitrogen starvation medium compared with rich medium, via10
did not show an increase of activity and via14 did not have Ape1p enzymatic activity under any condition (Fig. 7).

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Fig. 7.
Transport by autophagosomes does not restore
enzymatic activity. Cell lines were grown in nutrient-rich YPD
medium and nitrogen starvation MV-N medium to logarithmic
(L) and stationary (S) phases, and Ape1p
enzymatic activity was determined by the overlay assay. Images were
recorded with a CCD chemiluminescence camera system. The bottom
circle shows positions of the clones on the agar plates.
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Cvt Pathway as a Mechanism to Control pApe1p Proteolytic
Activity--
The Cvt pathway of aminopeptidase 1 is a nonclassic
pathway for the transport of vacuolar enzymes and vacuolar biogenesis, and it is not clear why such an additional pathway exists. The potentially harmful proteolytic activities of vacuolar enzymes are
controlled by their pro sequences and compartmentalization. Pro
sequences inhibit their enzymatic activities, and they are only
processed upon reaching low pH compartments as the endosome and
vacuole. In contrast the proteolytic activity of the cytoplasmic proteasome is controlled by chaperones and by selection and
modification of the proteins to be degraded by ubiquitination. Initial
studies on the four leucineaminopeptidases in Saccharomyces
cerevisiae indicated that pApe1p (or LAP IV) activity is also
regulated by processing through the vacuolar endopeptidase proteinase A
(21). Our data of the pApe1p transport mutants indicate that Cvt
vesicle formation controls enzymatic activity of the peptidase through the regulated dodecamerization and compartmentalization.
Dodecamerization is required for their enzymatic activity, and it
appears to be linked to Cvt vesicle formation. However, this would only
be a control mechanism, if the dodecameric precursor pApe1p already has
enzymatic activity.
The vacuolar endopeptidase proteinase A or PrA is required for the
maturation of vacuolar hydrolases and Cvt vesicles, and autophagosomes
are not degraded and accumulate as autophagic bodies within vacuoles
deficient in PrA. This leads to the accumulation of pApe1p in the
cells. We tested the
pep4 mutants of wt and via mutants for aminopeptidase 1 enzymatic activity (see
Fig. 3B). wt cells deficient for PrA had full aminopeptidase
1 enzymatic activity, whereas via10 and via14
were still deficient for aminopeptidase 1 activity, demonstrating that
the pro sequence of aminopeptidase 1 does not control the proteolytic
activity of the enzyme (Fig. 8).

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Fig. 8.
The precursor form pApe1p is proteolytically
active. wt and via mutants with and without proteinase
A ( pep4) were grown on minimal media for
16 h, and overlay assays for aminopeptidase 1 activity were
performed. Images were recorded with a CCD chemiluminescence camera
system.
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Vacuolar Function and Autophagy in via Mutants--
Accumulation
of cytoplasmic pApe1p Cvt transport vesicles could also be caused by
impaired vacuolar functions, and we therefore tested the mutants for
functional vacuoles. We first analyzed vacuolar morphology and
acidification by phase contrast microscopy and staining of the vacuoles
with the pH-sensitive fluorescent dye DC2LFDA (Fig.
9A). This did not reveal
differences in vacuolar morphology between the strains irrespective of
the growth stage or media (Fig. 9A shows cells from YPD at
A600 = 1), indicating that biogenesis of the
target organelle for pApe1p transport is not effected in the
mutants.

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Fig. 9.
A, acidic vacuoles in wt,
via10, and via14 visualized by the pH-sensitive
fluorescent dye DC2LFDA (right panel). Cells
shown were grown in YPD to A600 = 1, where the
Cvt pathway transport defect is observed. B, accumulation of
autophagic bodies in strains with deleted proteinase A gene
( pep4), but not in cells with active
proteinase A. Cells shown were grown in YPD to the stationary phase:
A, wt; B, wt pep4;
C, via10; D,
via10 pep4; E, via14; and
F, via14 pep4. Arrows
indicate autophagic bodies. C, growth of wt,
via14, and via10 strains in liquid nutrient-rich
YPD medium and of wt, via14, and via10 and these
strains with a deleted proteinase A gene
( pep4) in nitrogen-limited (MV-D)
medium and nitrogen starvation medium (MV-N) at 30 °C.
Liquid cultures were inoculated 1:10,000 from stationary YPD
cultures.
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Intact cell and vacuolar morphology of the via10 and
via14 mutants indicates that osmotic balance is not effected
in the mutants. To verify this effect, cell lines were grown in the
presence of 0.5, 1, and 1.5 M NaCl in YPD medium. wt and
both via strains displayed the same sensitivity to osmotic
stress and grew slower with increasing salt concentration up to 1.5 M (not shown). One known effect of osmotic imbalance on
membrane fusion events is the plasma membrane fusion reaction during
mating (24). Therefore, we quantified the mating reaction of
the via mutants. After 4 h, 10% of the cells in the wt
as well as the via mutants had completed the fusion reaction
as judged by the formation of fleurs-de-lille.
Many mutants of the Cvt pathway for aminopeptidase 1 are also defective
in autophagy and vice versa. Growing the mutants under autophagy-inducing conditions led to increased pApe1p transport to the
vacuole in both via mutants. This indicated specific defects in pApe1p transport along the Cvt pathway but not by autophagy. Therefore, we verified that autophagy is not impaired in via
mutants. Yeast deficient in the vacuolar endopeptidase PrA are unable
to degrade the vacuolar single membrane autophagic bodies. These accumulate within the vacuoles in these cells and can be seen in phase
contrast microscopy (22). Mutants deficient in autophagy and with a
deleted PrA gene do not accumulate autophagic bodies. The wt strain,
via10 and via14 as well as the respective strains with a deleted PrA gene (
pep4) were grown in
rich medium (YPD) to the stationary phase (Fig. 9B).
Autophagic bodies could be seen in via10
pep4
(D) and via14
pep4 (F) as
well as in the wt
pep4 (B) but not
in the strains with PrA activity (A, C, and
E in Fig. 9B). This demonstrates vacuolar
transport of autophagosomes in via10 and
via14.
Do the autophagosomes transport cargo proteins? Vacuolar proteinase PrA
is required for the maturation of several vacuolar hydrolases, and its
deficiency leads to impaired vacuolar function and reduced vitality of
PrA mutant strains under conditions of nutrient starvation (25).
Mutants of the Cvt pathway and autophagy have a reduced viability under
nitrogen starvation due to a block in cytoplasm to vacuole transport of
proteins to be degraded by macro-autophagy. Vacuolar protein turnover
and synthesis of vacuolar enzymes as well as proteins related to
vacuolar function are induced in cells grown in rich media (YPD) to
stationary phase (A600 > 2, glucose 0%) and to
a larger extend in nitrogen-limited media (MV-D) and nitrogen
starvation media (MV-N) (26). We tested via10 and
via14 for defects in the transport of cytoplasmic proteins to the vacuole by following growth in these media and tested for enhancement of stress under starvation conditions by deletion of the
PrA gene PEP4. Both via mutants grew as fast as
the wt strain and reached the same cell densities in rich media, but the via14 mutant showed a prolonged lag-phase after
inoculating the media from a G1-arrested stationary
culture in rich media (YPD) (Fig. 9C). The same result was
obtained when strains were grown in rich media containing ethanol as
the sole carbon source (YPE) (not shown). Under nitrogen-limiting
conditions in MV-D, neither of the via mutants reached the
cell densities of the wt strain (Fig. 9C), but
via10 cells grew in the early log-phase as fast as the wt
strain. via14 cells displayed a prolonged lag-phase also
under these conditions, grew slower then the wt and the
via10 cells, and did not reach the cell density of the
via10 mutant. Both mutant cultures contained 20% dead cells
compared with 6% in the wt culture during mid-log-phase at glucose
concentrations between 1.5 and 0.5 and 40% compared with 15% in
stationary cultures at 0% glucose as measured by trypan blue staining.
The increased sensitivity of the mutants to nitrogen starvation was
verified by growing the cells in nitrogen starvation medium (MV-N).
via14 was also able to grow under these conditions albeit at
a reduced rate. Deletion of the vacuolar PrA prolonged the lag-phase
after inoculating the cultures, but reduced the growth rates of the wt
pep4, via10
pep4 as
well as via14
pep4 mutants to the same extent.
The enhancement of the via mutant growth phenotype by PrA
deletion indicates that via mutants are not defective in
autophagocytic mechanisms and that the observed autophagic bodies
contain cargo proteins delivered to the vacuole for degradation as well
as pApe1p. However, the via mutants are stressed by
nutrient starvation indicating that, besides pApe1p and
-mannosidase, other proteins are transported along the Cvt pathway
as well.
 |
DISCUSSION |
pApe1p rapidly assembles with a half time of 2 min into a
dodecameric complex in the cytoplasm, which is packaged into
double-membrane Cvt vesicles. All pApe1p and mApe1p are in dodecameric
complexes (27). Targeting of pApe1p into Cvt vesicles requires the
propeptide, which consists of 45 amino acids forming a helix-turn-helix
structure. Mutations of the signal sequence, which prevent helix
formation, also abolish pApe1p transport but appear not to influence
dodecamer formation (15, 16). The via mutants of pApe1p
transport were isolated based on the absence of enzymatic activity,
which requires homododecamerization of the protein. In the two
complementation groups via10 and via14 pApe1p
does not form homododecamers but is transported into Cvt vesicles,
where it accumulates. The fraction of Ape1p transported to the vacuole
is present as a homododecameric complex. The Ape1p in the mutants is
sensitive toward cytoplasmic proteolysis leading to reduced pApe1p
levels under conditions of Cvt vesicle dependent transport.
To test for Cvt vesicle formation, the propeptide of pApe1p fused to
GFP was expressed under the APE1 promoter. This fusion protein is rapidly degraded in the cytoplasm as well as vacuoles and
can therefore be used to test for Cvt vesicle formation and to
demonstrate accumulated Cvt vesicles by immunofluorescence microscopy.
We could not detect immunofluorescence in the cytoplasm of the
via mutants. pro-GFP was degraded in all cell lines, but via mutants contained reduced amounts of the protein
demonstrating impaired Cvt vesicle formation.
Transport of pApe1p to the vacuole can be up-regulated in the mutants
by inducing autophagy, but this does not restore enzymatic activity and
thus homododecamerization. This demonstrates a specific defect in the
Cvt pathway and that dodecamerization of pApe1p is linked to the Cvt
pathway. This indicates that VIA10 and VIA14 encode novel factors with chaperone-like activities required for the
formation of native pApe1p homododecamers and transport by the Cvt
pathway and that these factors are also required for the formation of
native complexes under conditions of autophagy, but that they are not
required for vacuolar transport of autophagosomes. The cytoplasmic
HSC70 family members Ssa1p, Ssa2p, Ssa3p, and Ssa4p are required for
vacuolar transport of pApe1p by the Cvt pathway. HSC70 mutants
accumulate pApe1p in Cvt vesicles, indicating that they are required
for Cvt vesicle formation. Dodecamerization of pApe1p is not disturbed,
and we do not find evidence for a direct HSC70·pApe1p
interaction in vivo (18). We also expressed either
Ssa1p or Ssa2p from single-copy vectors in both via mutants, but this did not restore Ape1p enzymatic activity or
dodecamerization.2
How are dodecamerization and Cvt vesicle formation linked to each
other? (i) Is no dodecamer formed if no Cvt vesicles are present? (ii)
Are no Cvt vesicles formed without formation of dodecamers? or (iii) Is
the same protein or protein complex required for dodecamerization as
well as Cvt vesicle formation? These three questions are answered as
follows. (i) This is likely to be the case, because pApe1p
complexes accumulating upon overexpression of pApe1p lead to the
formation of oligomers, which are not transported to the vacuole (2).
(ii) Expression of pro-GFP, which does not oligomerize, is transported
by the Cvt pathway demonstrating that dodecamerization is not a
prerequisite for Cvt vesicle formation (23). (iii) Pro-GFP is not
protected by a Cvt vesicle membrane in the via mutants,
demonstrating that Cvt vesicle formation is disturbed in the mutants.
Dodecamerization of pApe1p in the cytoplasm is required for the
formation of enzymatically active dodecamers of aminopeptidase 1, because inducing autophagy restores pApe1p transport to the vacuole and
its proteolytic processing, but this does not restore enzymatic
activity. Therefore, we think that VIA10 and
VIA14 are required for the controlled dodecamerization as
well as Cvt vesicle formation, possibly as part of a multimeric complex. This way they can control the potentially harmful cytoplasmic proteolytic activity of pApe1p, because enzymatically active complexes are only formed, if they are readily compartmentalized. This method of
controlling proteolytic activity also explains the existence of a
second transport pathway for vacuolar enzymes.