From the Department of Biology, University of Michigan, Ann Arbor, Michigan 48109
Received for publication, February 6, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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One challenge facing eukaryotic cells is the
post-translational import of proteins into organelles. This problem is
exacerbated when the proteins assemble into large complexes.
Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome
in the yeast Saccharomyces cerevisiae. The precursor form
of API assembles into a dodecamer in the cytosol and maintains this
oligomeric form during the import process. Vacuolar delivery of the
precursor form of API requires a vesicular mechanism termed the
cytoplasm to vacuole targeting (Cvt) pathway. Many components of the
Cvt pathway are also used in the degradative autophagy pathway.
One of the hallmarks of eukaryotic cells is the presence of
a variety of membrane enclosed organelles. These compartments are
critical to the proper physiology of the cell. In order to ensure
correct function, cells must maintain faithful sorting of resident
proteins to each organelle. A substantial amount of information is
known about the delivery of proteins to compartments of the
endomembrane system including the endoplasmic reticulum (ER),1 Golgi complex, and
vacuole/lysosome. In brief, proteins can enter the ER
post-translationally through proteinaceous channels and transit to
other compartments through transient carrier vesicles. However, other
compartments are not able to receive proteins through this route. For
example, peroxisomes, mitochondria, and chloroplasts maintain a
separate mechanism(s) for translocating proteins across their membranes
(1-3).
One challenge facing eukaryotic cells is the import of large assembled
protein complexes into organelles. Although several organelles have the
capacity to translocate folded proteins (4), in most cases complexes
assemble subsequent to import of the individual protein monomers. In
contrast, the peroxisome has the capacity for importing oligomeric
proteins directly from the cytosol (5). Several components in
peroxisome protein import are currently being characterized, but the
actual mechanics of import are not defined (3). Both a transient
pore/shuttle or a vesicular invagination of the peroxisome membrane
could act in conjunction with the known receptor proteins. Although
most vacuolar proteins enter the vacuole through a portion of the
secretory pathway, the resident hydrolase aminopeptidase I (API) uses
an alternative process (6). Precursor API (prAPI) is synthesized in the
cytosol on ribosomes that are not attached to the ER. Following
synthesis, prAPI rapidly assembles into a dodecamer and subsequently
into a higher order complex (7). This complex of dodecamers becomes
sequestered by membrane, resulting in the formation of a
double-membrane cytosolic vesicle (8, 9). Upon completion, this vesicle
fuses with the vacuole to release a single-membrane vesicle into the
lumen. Following breakdown of the vesicle, prAPI is matured by removal
of its propeptide. This import process is termed the cytoplasm to
vacuole targeting (Cvt) pathway. The Cvt pathway has been shown to
overlap with autophagy (10, 11). Autophagy is a degradative process
that employs similar changes in membrane topology to sequester cytosol for breakdown and recycling under starvation conditions. During starvation, the level of prAPI increases and the protein is imported to
the vacuole via autophagosomes (9). An intriguing question has been why
yeast cells have utilized such a complex process as the Cvt pathway for
the import of a resident vacuolar hydrolase. One possibility is that
the dodecameric structure of prAPI is critical for its stability and/or
function. The size of the dodecamer prevents translocation through the
ER translocon, necessitating a vesicle-mediated import process. In
addition, it has not been known whether other resident hydrolases
utilize the Cvt pathway for vacuolar localization.
Reagents--
Prestained molecular weight markers were from
Bio-Rad. Oligonucleotide primers were synthesized by Operon
Technologies (Alameda, CA). The pAM1 plasmid (13) was a gift from Drs.
Michael J. Kuranda and Phillips W. Robbins (Massachusetts Institute of
Technology, Cambridge, MA). Yeast nitrogen base (YNB) was from Difco.
YNB without copper ions was from BIO 101 (Vista, CA). Ficoll,
DEAE-dextran, and the Vistra ECF Western blotting reagents were from
Amersham Pharmacia Biotech. Restriction enzymes, ligase, and DNA
polymerase were from New England Biolabs (Beverly, MA). Yeast lytic
enzyme was from ICN Biomedicals (Aurora, OH). Complete EDTA-free
protease inhibitors were from Roche Molecular Biochemicals.
n-Octyl- Strains and Growth Conditions--
Strains used were
SEY6210 MAT
Strains were typically grown on SMD (0.67% YNB, 2% glucose). Strains
were also grown to a density of 0.5 A600 in SMD
and were resuspended and grown in SGd (0.67% YNB, 3% glycerol, 0.1%
glucose) for induction of Ams1 where indicated. Strains that were
induced with 10 or 50 µM copper sulfate were first
subcultured three times in SMD lacking copper ions. All media were
brought to pH 5.5 with 50 mM each MES and MOPS and
supplemented with required amino acids and vitamins.
Plasmids--
The copper-inducible AMS1 plasmids
pCuAMS1(414) and pCuAMS1(424) (pMUH25 and pMUH26, respectively) were
generated as follows. Oligonucleotide primers (AM1LOWSPE,
5'-CAAAAACTAGTAATTATGTCATC-3'; and AM1UP2,
5'-CGCTTGCTCGAGCTCCTCTATGTGGATAG-3') were used to amplify a truncated
AMS1 sequence from pAM1 (13). The 2.9-kb product was ligated
into pCR-Blunt (Invitrogen, Carlsbad, CA), resulting in a 6.4-kb
construct with flanking SpeI sites (pMUH18). pMUH18 was
digested with SpeI, and the resulting 2.9-kb fragment was
ligated in the correct orientation at the SpeI site of
plasmid pCu414 (provided by Dr. Dennis J. Thiele, University of
Michigan, Ann Arbor, MI; Ref. 18), resulting in a 7.8-kb plasmid
(pMUH21). To restore the full-length sequence of AMS1, the
AMS1 gene was amplified from the pAM1 plasmid (primers:
AM1LOWER, 5'-CCTAACTCGTTTAAGGGAGAC-3'; and AM1UPPER,
5'-CAGTGAGGGAGACAAACTCAG-3'). A 3.4-kb product was ligated into
pCR-Blunt to generate a 6.9-kb plasmid (pMUH10). The 3.5-kb
XhoI/SpeI fragment from pMUH10 was ligated into
the same sites of pRS423 (19) generating pMUH12. The carboxyl terminus of AMS1 was restored with the 525-base pair
SphI/XhoI fragment from pMUH12 ligated into
pMUH21 resulting in the centromeric (CEN) plasmid pCuAMS1(414)
(pMUH25). The multicopy (2µ) plasmid pCuAMS1(424) (pMUH26) was
generated by ligating the 2.9-kb SpeI fragment from pMUH18
into the SpeI site of pCu424 (18), resulting in an 8.5-kb plasmid (pMUH22). The carboxyl terminus of AMS1 was restored
as described for pMUH25.
pCuAMS1YFP (pMUH29) was generated from pMUH21 and the 780-base pair
fragment from pEYFP (CLONTECH, Palo Alto, CA), each
digested with SphI/EcoRI. The resulting fusion
protein contained the amino-terminal 929 amino acid residues from Ams1
followed by a 14-amino acid linker and YFP sequence.
The ams1
The plasmid pCYI-50 (20) encodes the first 50 amino acids of
carboxypeptidase Y (CPY) including the vacuolar targeting signal, fused
to the periplasmic protein invertase, lacking its amino-terminal signal
sequence in the vector pSEYC306. The plasmid pLJL2 is similar to
pCYI-50 but carries the LEU2 gene instead of
URA3.
Vacuole Preparations--
Vacuoles were isolated on a Ficoll
step gradient essentially as described previously (21) with minor
adaptations. Cells were grown in SMD to early log phase, and then
~500 A600 units were harvested and
spheroplasted with 6-10 mg of yeast lytic enzyme. Ninety percent or
greater spheroplasting efficiency was determined by comparing the
A600 of cells diluted 1:10 in water before and after addition of lytic enzyme. The plasma membrane was lysed using 400 µl of a 0.4 mg/ml solution of DEAE-dextran by being chilled on ice
for 2 min, shaken at 30 °C for 2.5 min, and placed on ice again. 3 ml of lysed cell solution containing 2 mM
phenylmethylsulfonyl fluoride were loaded at the bottom of an
ultraclear SW41 tube (Beckman Coulter, Fullerton, CA) and overlaid with
8%, 4%, and 0% Ficoll solutions. Vacuoles were collected from the
0%/4% float interface with a Pasteur pipette after a 1.5-h spin at
30,000 × g. Enzyme assays for marker proteins were
performed on material loaded onto the gradient and the recovered
vacuole float fraction.
Enzyme Assays--
Cell lysates and harvested vacuoles were
assayed for marker enzymes invertase, Immunoblotting and Antisera--
Antisera was generated to Ams1
using two synthetic peptides (Multiple Peptide Systems, San Diego, CA)
corresponding to amino acid residues 79-96 and residues 991-1011.
These peptides were conjugated to keyhole limpet hemocyanin and
injected into a New Zealand White male rabbit using standard
procedures. Immunoblots were prepared from 8% SDS-PAGE gels as
described (16) with 15% methanol in the transfer buffer. Ams1
antiserum was blocked by incubating with ams1 Glycerol Gradient--
Strains harboring the pCuAMS1(414)
plasmid encoding AMS1 under a copper regulable promoter were
grown in SMD in the absence of copper. The presence of this plasmid
facilitated the visualization of Ams1 under the indicated growth
conditions. Cells were induced with 50 µM
CuSO4 for 1 h. Twenty A600
units were harvested in log phase and washed in Tris salts buffer (TSB;
50 mM Tris, pH 8.5, 50 mM KOAc, 100 mM KCl, 0.5 mM MgCl2). Cells were
transferred to a microcentrifuge tube and resuspended in 250 µl of
TSB containing 1 µg/ml pepstatin A and Complete EDTA-free protease
inhibitors. Cells were lysed with glass beads for 1 min, and then
Ten supernatant fractions of 200 µl were collected from the top of
the gradient and precipitated with 10% trichloroacetic acid on ice.
Fraction 10 may contain some amount of contaminating pellet material.
Samples were washed twice in acetone and sonicated in MES/urea
resuspension buffer (6) before resolving by SDS-PAGE, followed by
immunoblot and detection of Ams1 or API. A higher molecular mass
cross-reactive protein detected by Ams1 antiserum (see Fig.
1B, lane 5) migrated to fractions 3-4
in wild type and cvt2 cells (Fig. 3, A and
B). Quantification of these fractions was arbitrarily set to
zero for clarity. Protein standards were run on an identical gradient
and the peak fraction for each was determined by Coomassie Brilliant
Blue staining of a SDS-PAGE gel. Bovine serum albumin (66 kDa),
aldolase (158 kDa), and catalase (240 kDa), peaked in fractions 3, 4, and 5, respectively, whereas apoferritin (450 kDa), urease (545 kDa)
and thyroglobulin (669 kDa) all peaked in fraction 6 of the gradient.
For separation of the MHY11 vacuole fraction on a glycerol gradient,
purified vacuoles were collected as described above and 10× TSB was
added to 1× prior to a 5-min, 4 °C centrifugation step at 13,000 rpm. The pellet containing the concentrated vacuoles was resuspended in
250 µl of TSB plus inhibitors and detergent and mixed, and 200 µl
was loaded onto the gradient and collected as above.
Fluorescence Microscopy--
Confocal microscopy (Leica IRM
confocal microscope) images were taken as an average of 4-8 scans of a
single focal plane. Cells harboring the pCuAMS1YFP plasmid (pMUH29)
were grown to early log phase and induced with 10 µM
copper sulfate for 12 h. 1 ml of cells were then harvested and
resuspended with 100 µl of fresh medium containing 4-8
µM FM 4-64 (23) for 30 min to label the vacuoles. FM
4-64 was chased to the vacuole for 1 h by the addition of 1 ml of
fresh medium. Cells were then washed once in SD Ams1 Delivery to the Vacuole under Vegetative Conditions Is
Dependent on Machinery of the Cvt and Autophagy Pathways--
Most of
the characterized vacuolar hydrolases are proteolytically processed
upon delivery to the vacuole. The resulting shift in molecular mass
provides a convenient means for monitoring delivery to the organelle.
We were interested in determining the mechanism of vacuolar delivery
used by the resident hydrolase
Ams1 has a predicted molecular mass of 124 kDa. Previous studies
indicated that the initial protein product was 107 kDa and that this
was processed to 73 and 31 kDa forms that co-purified (24). However, we
detected a band corresponding to Ams1 that migrated at 122 kDa (Fig.
1B) that is closer in agreement with the expected molecular
mass and probably corresponds with the full-length protein. To increase
the level of Ams1, we expressed the protein from a CEN plasmid under
control of the CUP1 promoter. A 73-kDa cleavage product was
not detected in wild type cells grown in SMD (Fig. 1B,
lane 1). After cells were grown to early log
phase and shifted to glycerol medium (SGd), a 73-kDa protein was
apparent that first appeared ~12 h after the shift. This band increased in abundance from 12 to 24 h (Fig. 1B,
lanes 2 and 3). Appearance of the
73-kDa species was not seen in a pep4
Because Ams1 is not processed concomitant with its delivery to the
vacuole, we could not rely on a mobility shift to follow localization.
As an alternative approach, we analyzed its localization through
subcellular fractionation. Yeast cells were grown to early log phase in
minimal medium, and vacuoles were isolated on a Ficoll step gradient as
described under "Experimental Procedures." The efficiency of
vacuolar recovery was determined by assessing the invertase activity of
a vacuolar targeted fusion protein consisting of a portion of the
resident vacuolar protease CPY fused to the marker protein invertase
(20). This chimeric construct utilizes the vacuolar targeting signal in
CPY to divert the periplasmic enzyme invertase from the secretory
pathway to the vacuole. The percentage of invertase activity in the
purified vacuole fraction relative to the activity in a total
spheroplast lysate provides a measure for the efficiency of organelle
purification. Vacuole recovery of the CPY-invertase hybrid protein
typically was 20% or higher as compared with the activity in the total
fraction. In wild type cells, Ams1 activity in the purified vacuolar
fraction was essentially equivalent to the vacuolar recovery based on
invertase activity from the CPY-invertase marker (Fig.
2A). This result indicates
that vacuole purification is an effective method for monitoring
vacuolar delivery of Ams1.
We next examined whether Ams1 was delivered to the vacuole in strains
defective specifically in the Cvt pathway. The cvt3 and
cvt9 mutants accumulate the precursor form of API but are relatively normal for autophagy (11, 25). The Cvt9 protein interacts
with the Apg1 kinase and may be part of a complex that includes Apg13,
Apg17, and Vac8 (25-27). The CVT3 gene has not been cloned,
but the cvt3 mutant appears to be defective in vesicle formation/completion because prAPI is protease-sensitive in this strain
(16). The percent recovery of CPY-invertase was comparable in wild type
and mutant strains, indicating that its delivery to the vacuole was not
affected by cvt mutations (Fig. 2A). Accordingly, CPY-invertase is a useful marker for comparison to Ams1. Total Ams1
activity for wild type and mutant strains was comparable. However, the
Ams1 activity in the vacuolar fraction of the cvt3 and
cvt9 mutants was negligible, indicating that Cvt components are required for the transport of Ams1 to the vacuole (Fig.
2A). Contamination of the vacuolar fraction by other
organelles or cytosol was determined by assaying the purified vacuole
fraction for NAPDH-cytochrome c reductase activity (ER
marker) and
We next examined mutants that are defective both in autophagy and the
Cvt pathway. We decided to include in the analysis mutants that were
blocked at different stages of the Cvt and autophagy (Apg) pathways
(Table I). For example, the Apg1 kinase
appears to be part of a complex that may be involved in regulating the conversion between the Cvt and Apg pathways (25, 27). Apg7 is a homolog
of the E1 ubiquitin activating enzyme (28, 29). This protein is
required for the conjugation of Apg12 to Apg5 and also for the addition
of phosphatidylethanolamine to Aut7 (30, 31). The Aut7 protein is the
only characterized component of these pathways that shows a substantial
up-regulation of synthesis under autophagy-inducing conditions (32,
33). Aut7 is required for completion of Cvt vesicles and for expansion
of the autophagosome membrane (34). Finally, Cvt17 is required for the
breakdown of subvacuolar vesicles. The Cvt and autophagy pathways
utilize double-membrane cytosolic vesicles to sequester cytoplasmic
constituents. Fusion of these vesicles with the vacuole results in the
release of a single-membrane subvacuolar vesicle, either a Cvt or
autophagic body depending on the nutrient conditions. These subvacuolar
vesicles are subsequently degraded within the vacuole lumen in a
process that is dependent on Cvt17 and the vacuolar hydrolase
proteinase B (35, 36).
The Ams1 activity recovered in vacuole fractions of most
apg, aut, and cvt mutants was again
negligible (Fig. 2B). As expected, the one exception was the
cvt17 mutant (Fig. 2B). The recovery of vacuolar
Ams1 activity in cvt17 was similar to that of CPY-invertase, indicating that Ams1 was located within vacuoles in the
cvt17 strain, presumably within Cvt bodies. Similar results
were seen with a pep4 Ams1 Is Imported as an Oligomer--
Precursor API is oligomerized
in the cytosol to form a dodecamer (7). The dodecameric form is
maintained during the import process. The dodecameric precursor is
~732 kDa in mass and is too large to translocate through a
proteinaceous channel such as the ER translocon. Accordingly, the
particular biosynthesis of prAPI necessitates the use of a
vesicle-mediated import mechanism such as the Cvt pathway. Because the
cvt mutants lacked the ability to import Ams1, we were
interested to see if oligomerization was a characteristic of this
second cargo protein.
To determine whether oligomerization of Ams1 took place prior to
import, wild type and mutant strains were analyzed by glycerol density
gradients (Fig. 3). Protein extracts were
prepared under native conditions and separated on a 20-50% glycerol
density gradient in the presence of
We next wanted to determine if Ams1 was present as an oligomer in the
cytosol and if so, whether it retained its oligomeric structure during
import into the vacuole. Accordingly, we examined apg and
cvt mutant strains to determine the oligomeric state prior to and following sequestration by Cvt vesicles. As noted above, the
apg7/cvt2 mutant is defective in the function of an E1-like protein (28, 29) and is required for both the Cvt and Apg pathways. The
apg7 mutant is blocked at the vesicle formation/completion step and accumulates prAPI in a membrane-bound form that is not within
a completely enclosed vesicle. Ams1 peaked at fraction 6 of the
glycerol gradient in the apg7 mutant (Fig. 3B).
Because this mutant is blocked in vacuolar delivery of Ams1 (Fig.
2B), these data suggest that Ams1 oligomerizes in the
cytosol. As discussed above, the cvt17 strain is defective
in the breakdown of subvacuolar vesicles that result from the Cvt and
Apg pathways. Ams1 was found to exist as an oligomer in the
cvt17 strain (Fig. 3C). The appearance of
oligomeric Ams1 in the cytosol and in subvacuolar vesicles suggests
that it maintains its oligomeric state during the import process.
Ams1 Arrives in the Vacuole by the Autophagy Pathway under
Starvation Conditions--
Precursor API is delivered to the vacuole
by the Cvt or autophagy pathway depending on the nutrient conditions
(9). To examine the localization of Ams1 under autophagic conditions, we followed the protein in vivo by confocal microscopy. Ams1
was placed under the control of a copper-inducible promoter and fused to YFP as described under "Experimental Procedures." Yeast cells were labeled with the dye FM 4-64 to mark the vacuole (23) and incubated under nitrogen starvation conditions (SD Ams1 Is a Second Cargo Protein of the Cvt Pathway--
Resident
hydrolases of the vacuole/lysosome typically transit to the vacuole
through a portion of the secretory pathway during biosynthesis. An
alternate route, the Cvt pathway, is utilized by the vacuolar hydrolase
API. The distinct characteristic of this biosynthetic route is the
formation of double-membrane vesicles that sequester dodecameric prAPI
in the cytosol. Under starvation conditions, this large oligomeric
protein complex is taken to the vacuole via autophagy, which is
morphologically and mechanistically similar to the Cvt pathway. Common
gene products are involved in Cvt vesicle/autophagosome formation,
vesicle fusion to the vacuole, and breakdown of vesicles delivered to
the vacuole (for a detailed review, see Ref. 37). Many of these
components act prior to vesicle formation such as Apg1, Apg7, Apg9, and
Apg14 (15, 27, 28, 33, 38). Aut7 also acts at the stage of vesicle
formation/completion but, unlike these other components, travels to the
vacuole inside of the vesicles (31, 39). Finally, Cvt17 and Prb1 are
critical for breakdown of the subvacuolar vesicles (35, 36). Opposing
features of the selective Cvt pathway and non-selective autophagy occur
at points of regulation, specificity, and cargo capacity. Some
components required for delivery of proteins from the cytoplasm to the
vacuole are specifically required for the Cvt pathway and others for
autophagy. For example, selective vesicular targeting by the Cvt
pathway depends on Cvt3, Cvt9, and Vac8 (9, 25, 26), whereas Apg17 is
only needed for autophagy (27). Until now, prAPI was the only specific
cargo protein that was definitively shown to use the Cvt pathway. In this study, we have identified a second cargo protein of the Cvt pathway,
Ams1 has been the canonical vacuole/lysosome membrane marker protein
(40), but, until now, its mode of import in Saccharomyces cerevisiae was unclear. Like API, Ams1 is not glycosylated, nor does it utilize the secretory pathway (12). A decade has passed since
researchers first attempted to define the import mechanism for Ams1.
This is in part due to the difficulty of working with this protein.
Ams1 is made at low levels during vegetative growth. In addition, prAPI
undergoes proteolytic removal of its amino-terminal propeptide region
coincident with vacuole import and enzyme activation. In contrast, a
kinetically relevant processing event for Ams1 is not discernible, nor
is the activation of Ams1 dependent on vacuole localization (12).
Previously, import kinetics for Ams1 were assessed by its
Pep4-dependent cleavage (12). In that study, cells were
stressed by heat shock and then radiolabeled at 37 °C. Under these
conditions, the half-time of delivery was determined to be ~10 h.
However, the cleaved protein product was not localized subcellularly.
This time frame also does not satisfy basal transport requirements of a
cell that is growing with a 1.5-h doubling rate, as is the case in SMD
. This Pep4-dependent cleavage event, therefore, does not
appear to be a valid way to monitor vacuole delivery for Ams1. In fact,
protease-susceptible sites present within Ams1 result in a 73-kDa
polypeptide upon the addition of exogenous protease (12) or under
environmental stress conditions (Fig. 1B). It is currently
unclear whether there is a physiological significance to the
proteolytic conversion of the 122-kDa Ams1 to the 73-and 31-kDa forms.
In contrast to previous studies, we conclude that Ams1 is not cleaved
to a mature form directly upon delivery to the vacuole. Because
proteolytic processing does not appear to occur coincident with
vacuolar delivery, it is problematic to determine the kinetics of
import for Ams1. We speculate that the half-time for delivery of Ams1
is similar to that of prAPI (20-30 min; Ref. 11) because of the
dependence on the selective vacuolar targeting gene products such as
Cvt3 and Cvt9 (Fig. 2A). Due to the absence of a convenient gel mobility assay to follow localization, we relied on subcellular fractionation to monitor vacuolar Ams1 delivery. All cvt,
apg, and aut mutants examined, with the exception
of cvt17, were found to be defective in vacuolar delivery of
Ams1 (Fig. 2). The recovery of Ams1 in the vacuoles of the
cvt17 strain under vegetative conditions is consistent with
the premise that Ams1 is a Cvt cargo protein. Subvacuolar vesicles
containing prAPI have been shown to accumulate in the cvt17
mutant as well as in a pep4
Recently, we have characterized the Cvt19 protein as a receptor for
resident vacuolar hydrolases that utilize the Cvt
pathway.2 Targeting of prAPI
under starvation conditions requires Cvt19 even though nonspecific
autophagy is functional in the cvt19 Ams1 Is Imported into the Vacuole as an Oligomer--
Gel
filtration chromatography has shown that vacuolar Ams1 is a 560-kDa
hetero-oligomer (24). Although a 107-kDa form was only reported from
pep4
Crossing a membrane as an intact oligomeric complex is a major
challenge. Dodecameric prAPI is ~732 kDa in mass, too large to
translocate through a proteinaceous channel such as that present in the
ER. Accordingly, prAPI is sequestered within vesicles during their
formation in the cytosol. Similarly, Ams1 uses this pathway for
vacuolar localization. There are two ways to interpret the oligomeric
assembly data. Precursor API appears to be unstable as a
monomer.3 Vacuolar proteins
that need to exist as oligomers either for stability or function may
need to utilize the Cvt pathway for localization. Conversely, it is
possible that these proteins form oligomers so that they are imported
more efficiently by the Cvt pathway and autophagy to satisfy other
cellular demands. For example, both Ams1 and API are up-regulated
during starvation. Macromolecular turnover is essential under these
conditions. By coupling autophagy with the import of certain key
hydrolases, the cell ensures the presence of both substrates and
degradative enzymes within the vacuole. There is an apparent redundancy
of hydrolases within the vacuole including carboxypeptidases S and Y,
aminopeptidases I and Y, and proteinases A and B. The Cvt pathway
presents an alternative mechanism for delivering hydrolases to the
vacuole, ensuring the presence of at least minimal degradative capacity if the Vps route to the vacuole is defective. There are additional reasons for Ams1 to utilize the Cvt pathway. Ams1 is
membrane-associated but is not an integral membrane protein. It lacks a
signal sequence or hydrophobic domain that could act as an internal
uncleaved signal sequence so that it is unable to enter the secretory
pathway. Finally, Ams1 is a mannose glycosidase that appears to be
constitutively active. It would be problematic for this enzyme to
traverse the secretory pathway along with mannosylated glycoproteins.
Ams1 Utilizes the Autophagy Pathway for Vacuolar Delivery under
Starvation Conditions--
Environmental stress conditions such as
starvation signal a major change in yeast physiology. Although the Cvt
pathway exists primarily as a biosynthetic route to the vacuole,
autophagy is capable of delivering both resident vacuolar hydrolases
and bulk quantities of their substrates to this organelle. It is
therefore reasonable to expect autophagy to carry Ams1 into the vacuole during nutrient limitation. We investigated the localization of an
Ams1YFP fusion protein under starvation conditions. Both
cvt17 and pep4
The importance of understanding the molecular mechanisms of protein
transport in the lower eukaryotes is becoming more evident as human
genome data become available. Although it has not yet been
characterized, the human homologue to AMS1 was recently
isolated from cDNA (GenBank accession no. AF044414). The highly
homologous encoded protein (35% identical, 51% similar to Ams1
according to BLAST results; Ref. 41) is more closely related to
the isolated rat -Mannosidase (Ams1) is another resident hydrolase that enters the
vacuole independent of the secretory pathway; however, its mechanism of
vacuolar delivery has not been established. We show vacuolar
localization of Ams1 is blocked in mutants that are defective in the
Cvt and autophagy pathways. We have found that Ams1 forms an oligomer
in the cytoplasm. The oligomeric form of Ams1 is also detected in
subvacuolar vesicles in strains that are blocked in vesicle breakdown,
indicating that it retains its oligomeric form during the import
process. These results identify Ams1 as a second biosynthetic cargo
protein of the Cvt and autophagy pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Mannosidase (Ams1) is a resident vacuolar hydrolase that has been
shown to enter the vacuole independent of the secretory pathway (12).
As a resident vacuolar hydrolase, one role of Ams1 is to aid in
recycling macromolecular components of the cell through hydrolysis of
terminal, non-reducing
-D-mannose residues (EC
3.2.1.24). In growing wild type cells, a relatively low basal level of
Ams1 is synthesized. Under conditions of greater recycling need
(i.e. nutrient deprivation), Ams1 levels are induced, and
this coincides with induction of the nonspecific delivery mechanism of
autophagy. We show in this report that Ams1 forms an oligomer in the
cytoplasm. Under nutrient-rich conditions, oligomeric Ams1 is delivered
to the vacuole by the Cvt pathway. Under starvation conditions, the
up-regulated oligomeric protein is localized through autophagy. Mutants
in either pathway are defective in Ams1 import. These results define
Ams1 as a second cargo protein that utilizes both the Cvt and
autophagic pathways for biosynthetic delivery to the vacuole.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside detergent (
OG)
was from Calbiochem (La Jolla, CA).
N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 4-64) was from Molecular Probes (Eugene, OR).
Polyclonal antiserum to the HA epitope was from Santa Cruz
Biotechnology (Santa Cruz, CA). Other reagents were from Sigma unless noted.
ura3-52 leu2-3,112
his3-
200 trp1-
901 lys2-801
suc2-
9 mel GAL (14); JKY007, SEY6210
agp9
(15); AHY1468, SEY6210 apg1/cvt10-4;
THY193, SEY6210 apg7/cvt2-1; AHY1293, SEY6210
apg14/cvt12-1; THY313, SEY6210 aut7/cvt5-1;
THY119, SEY6210 cvt3-1; AHY96, SEY6210 cvt9-1;
THY32, SEY6210 cvt17-1 (10, 16). MHY10 (SEY6210
pep4
::URA3) was generated as
described for MHY8 (17). THY411 (SEY6210
ams1
::LEU2) was generated by transforming yeast with PvuII-linearized pDelLAMS (described
below), which replaces the first third of the chromosomal
AMS1 gene with LEU2. The 3×HA-tagged Ams1 strain
MHY11 was generated by the integration of a DNA fragment encoding the
3×HA epitope at the 3' end of the chromosomal AMS1 locus in
strain SEY6210 using the ME3 plasmid (provided by Dr. Neta Dean, State
University of New York, Stony Brook, NY) as a template (primers:
AMSPREHA
(5'-AATTGAGACCTTTTGAGATTGCCTCATTCAGGTTGTATTTCTACCCATACGATGTTCCT-3') and
AMSpastHA
(5'-TTTACTTATATGTATTTTGTTAAGACTATTTTTGGTTATCAGTCGACGGTATCGATAAG-3')). Confirmation of the integrated
AMS1::AMS1-HA was shown by induction of
a ~132-kDa fusion protein. Integration of the HA epitope at the
chromosomal AMS1 locus had no detectable effect on Ams1
activity or its vacuolar localization (data not shown).
::LEU2 deletion plasmid,
pDelLAMS1, was generated from a 1-kb upstream portion and a 1.5-kb
interior portion of the AMS1 gene isolated separately from
pAM1 by a XhoI/HindIII digest and a
BglII/EcoRI/HindIII digest,
respectively. These fragments were ligated in the vector pRS306 (19)
resulting in a unique HindIII site between the two segments
of AMS1. This plasmid was digested with HindIII,
and a HindIII fragment containing the LEU2 gene
was cloned into this site to generate plasmid pDelLAMS1.
-glucosidase, and
NADPH-cytochrome c reductase as described (20). Ams1
activity was determined based on established protocol (22). Samples
were treated with Triton X-100 (2.5% final concentration), and then
the volume was brought up to 400 µl with distilled H2O.
100 µl of 5× substrate mix (200 mM sodium acetate, pH
6.5, 2 mM
p-nitrophenyl-
-D-mannopyranoside) was added to start the reaction and was incubated for 1 h at 37 °C. The reaction was stopped with 200 µl of 10% trichloroacetic acid, and
any particulates were spun down in a microcentrifuge for 5 min. An
equal volume of 1 M glycine, pH 10.4, was added to
neutralize the reaction before the absorbance at 400 nm was read.
Results from assays for each strain were tabulated from a minimum of
four independent vacuole preparations.
cell
extract in TTBS (20 mM Tris, pH 7.6, 0.8% NaCl,
0.1% (w/v) Tween 20) for 2-3 h at 4 °C prior to use at a 1:12,500
dilution for 4 h at room temperature. Polyclonal antiserum to the
HA epitope was used at a 1:1,000 dilution. API antiserum was described
previously (6). Quantitation by Vistra kit or use of secondary
antibodies conjugated to horseradish peroxidase was as described (16,
17). Immunoblots were quantified on a STORM PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
OG
was added to 2% final concentration and mixed gently with a pipette tip. 200 µl of sample was loaded on a 1.8-ml prepoured 20-50% glycerol gradient made up in TSB and containing protease inhibitors. Oligomeric complexes were resolved at 55,000 rpm for 4 h in a Beckman TLS-55 rotor at 15 °C.
N (containing 10 µM copper sulfate) and incubated in fresh SD
N for
12 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase (Ams1). However,
previous analyses of Ams1 have been equivocal with regard to whether
this protein is rapidly processed following delivery to the vacuole
(12, 24). To examine processing of Ams1, we generated antiserum to
synthetic peptides corresponding to the deduced amino acid sequence as
described under "Experimental Procedures." We were unable to detect
Ams1 synthesized from the chromosomal locus when cells were grown in
minimal medium (SMD). This result was not surprising because Ams1 is
made at very low levels in the presence of glucose unless cells are
subjected to heat shock (12, 22, 24). However, when Ams1 was
synthesized from a CEN or 2µ plasmid, we were able to detect a
specific band in a dosage-dependent manner (Fig.
1A).
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Fig. 1.
Biosynthesis of Ams1. A,
antiserum to Ams1 recognizes a 122-kDa band. Wild type (SEY6210;
lane 1), ams1 (lane
2), and wild type cells harboring AMS1 on a CEN
or 2µ plasmid (lanes 3 and 4) were
grown in SMD to the early log phase. Ams1 expressed from plasmids is
detected in a dosage-dependent manner. Plasmid-containing
strains were induced with 50 µM copper sulfate for 40 min
prior to harvest. Cells were lysed with glass beads and run on an 8%
SDS-PAGE gel for immunoblot detection using anti-Ams1 antiserum. 1.0 A600 equivalent units of extract was loaded in
lanes 1 and 2 and 0.25 units in
lanes 3 and 4. B,
Pep4-dependent Ams1 cleavage is not a direct indication of
vacuolar delivery. Steady-state levels of Ams1 are shown at various
conditions for several strains. Strains in lanes
1-4 contain the CEN AMS1 plasmid pCuAMS1(414).
Lane 1, wild type cells grown to early log phase
in SMD and induced for 40 min with 50 µM copper sulfate.
Lanes 2 and 3, wild type cells shifted
12 or 24 h to SGd (containing 10 µM copper sulfate),
respectively. The level of Ams1 is increased, and a 73-kDa cleavage
product (indicated by the asterisk) appears as cells are
deprived of glucose (lanes 2 and 3).
Lane 4, pep4
cells shifted 24 h to SGd (containing 10 µM copper sulfate). Lack of the
73-kDa species indicates this cleavage event is
Pep4-dependent. Lane 5,
ams1
cells shifted 24 h to SGd (containing 10 µM copper sulfate) is shown as a control for
cross-reactive protein recognized by the anti-Ams1 antiserum.
WT, wild type.
strain (Fig.
1B, lane 4), indicating that it was
due to a vacuole-dependent cleavage event in agreement with
previous studies (12). However, the slow kinetics of cleavage and the
observation that processing was incomplete, coupled with the fact that
this processing event is not required for Ams1 enzymatic activity (12),
suggest that the cleavage event may be a byproduct of vacuolar
localization. Accordingly, processing of Ams1 is not a good indicator
for a kinetic analysis of vacuolar delivery.
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Fig. 2.
Vacuole delivery of Ams1 is defective in
cvt, apg, and aut
mutants. A, mutants specific to the Cvt pathway
do not import Ams1 into the vacuole. Vacuoles were isolated on Ficoll
step gradients as described under "Experimental Procedures."
Activity assays of Ams1 and marker proteins show equivalent recovery of
Ams1 and CPY-invertase in the vacuole fraction of wild type
(WT) cells, but not in the vacuole fraction purified from
the cvt3 and cvt9 mutant strains. CPY-invertase
(vacuole marker), white bar; Ams1,
black bar; -glucosidase (cytosol marker),
light gray bar; NADPH-cytochrome
c reductase (ER marker), dark gray
bar. Vacuoles were purified at least four times for each
strain shown, and the total enzyme activity recovered in the vacuole
fraction was divided by the total activity loaded on the gradient to
obtain the percentage of recovery. Average recovery values are shown
with error calculated as the standard deviation. B, mutants
defective in both autophagy and the Cvt pathway are also defective in
vacuolar delivery of Ams1. Ams1 is not recovered in the vacuole
fractions from apg1, apg7, apg9
,
apg14, and aut7 strains. Percentage of recovery
was as determined for A. The cvt17 mutant
accumulates Cvt vesicles within the vacuole, and shows equivalent
vacuole recovery of Ams1 due to the accumulation of Ams1 within these
vesicles.
-glucosidase (cytosol marker), respectively (Fig.
2A). The low level of Ams1 recovered in the purified vacuole
fraction from most mutant strains was not substantially higher than the
level of contamination from cytoplasmic markers. These data indicate
Ams1 utilizes the Cvt pathway for vacuolar delivery under vegetative
growth conditions.
Characterized genes required for Ams1 targeting
mutant strain that accumulates
subvacuolar vesicles due to a defect in activation of proteinase B
(data not shown). The dependence of Ams1 localization on Cvt, Apg, and
Aut components suggests that Ams1 delivery to the vacuole requires both
the Cvt and Apg components under vegetative conditions, and represents the second biosynthetic cargo protein that utilizes the Cvt pathway for
vacuolar delivery.
OG detergent as described under
"Experimental Procedures." As a control in this experiment, we
analyzed the migration of API. The mature form of API peaked at
fraction 7 of the gradient in agreement with our previous studies,
indicating that it is present in the vacuole as a dodecamer (Fig.
3A; Ref. 7). Similarly, Ams1 from total cell lysates did not
migrate on the gradient at a size expected for a monomer. Ams1 peaked at fraction 6, which corresponded to a molecular mass of 450-669 kDa
based on marker protein analysis. The peptide antiserum to Ams1
cross-reacted with a diffuse background band (see Fig. 1B, lane 5) that migrated at fractions 3 and 4 of the
glycerol gradient. To verify that Ams1 was not migrating at this part
of the gradient, we examined the oligomeric state of the protein using
anti-HA serum and a strain where the chromosomal AMS1 locus
had been replaced with an HA-tagged AMS1 gene (see
"Experimental Procedures"). Again, we found that Ams1 peaked at
fraction 6 and no material was detected at the less dense region of the
gradient (Fig. 3A). Therefore, in wild type cells, Ams1
appears to exist as an oligomer composed of 4-6 of the 122-kDa
species.
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Fig. 3.
Ams1 transits to the vacuole as an
oligomer. A, wild type (WT) cells harboring
the CEN Ams1 plasmid pCuAMS1(414) were grown to early log phase and
induced with 50 µM copper sulfate for 1 h. Strain
MHY11 (AMS1::AMS1-HA) was grown to
early log phase and harvested directly. 20 A600
units of cells were lysed with glass beads, mixed with OG detergent
(2% final concentration), and loaded on a 20-50% glycerol gradient.
Ten fractions of 200 µl were collected from the top and examined by
immunoblot. Immunoblots were probed with anti-Ams1, anti-HA, or
anti-API antisera and then quantitated using the Vistra detection
reagents as described under "Experimental Procedures." Ams1 peaked
as an oligomeric complex in fraction 6, whereas mAPI peaked at fraction
7 (7). Protein standards run on an identical gradient included: bovine
serum albumin (66 kDa, fraction 3), aldolase (158 kDa, fraction 4),
catalase (240 kDa, fraction 5), apoferritin (450 kDa, fraction 6),
urease (545 kDa, fraction 6), and thyroglobulin (669 kDa, fraction 6).
Note that the diffuse band in fractions 3 and 4 of the gradient
detected with peptide antiserum is a cross-reacting contaminant and was
not included in the quantification. B and C,
oligomerization of Ams1 occurs in the cytosol and is maintained during
vacuolar delivery. Native protein extracts from the apg7 and
cvt17 mutant strains harboring pCuAMS1(414) were analyzed by
glycerol gradients as in A. Ams1 is an oligomer in
apg7, a mutant that is defective in vacuolar delivery of
Ams1, and in cvt17 that is defective in the breakdown of Cvt
bodies.
N) for 12 h to
induce autophagy and allow import of the hybrid protein. In SD
N,
Ams1YFP was seen accumulating in the subvacuolar vesicles of
cvt17 and pep4
strains (Fig.
4) consistent with the vacuole fractionation data (Fig. 2B, data not shown). The
apg1 and apg7 strains that are defective in
autophagy showed no subvacuolar accumulation of Ams1YFP (Fig. 4), nor
did strains that harbored a copper-inducible YFP construct alone (data
not shown). Appearance of Ams1YFP in the vacuole was dependent on
components of the autophagic pathway. These data demonstrate that Ams1
delivery to the vacuole was mediated by autophagosomes under conditions
that induce autophagy.
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Fig. 4.
Ams1 is delivered to the vacuole by
autophagy. The Ams1YFP fusion protein was expressed in
cvt17, pep4 , apg1, and
apg7 cells and visualized by confocal microscopy using the
GFP channel as described under "Experimental Procedures."
Vacuoles were labeled with FM 4-64, and cells were starved in
SD
N for 12 h. Ams1YFP accumulates in subvacuolar vesicles of
cvt17 and pep4
strains under nitrogen
starvation conditions. Ams1 is not delivered to the vacuole in
apg1 and apg7 strains that show only cytosolic
localization of Ams1YFP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase.
strain (9, 11).
strain. The
cvt19
mutant is defective in vacuolar localization of
Ams1.2 Hence, Cvt19 is required for the specific transport
of both cargo proteins through the Cvt pathway and at least for prAPI
by autophagy and may represent a limiting factor for import of these
resident hydrolases. These data support the conclusion that Ams1
utilizes the Cvt pathway.
cells (12), we observed a form migrating at 122 kDa
in wild type cells (Fig. 1). The 122-kDa mass is in closer agreement to
the predicted size of Ams1 based on its deduced amino acid sequence. In
the present study, we show that the 122-kDa form of Ams1 assembled into
an oligomeric conformation in the cytosol (Fig. 3B).
Furthermore, this state was maintained throughout vacuolar delivery
(Fig. 3C). It is apparent from the data presented here that
vacuolar Ams1 is most likely a homo-oligomer composed of 122-kDa
subunits. These results suggest that one interesting feature of the Cvt
pathway is that the cargo proteins, prAPI and Ams1, are imported as
oligomers (Ref. 7; Fig. 3C).
mutant strains are defective in
breakdown of subvacuolar vesicles, independent of their origin (9, 35).
In addition to localizing Ams1 activity to the vacuole fraction under
vegetative conditions (Fig. 2B and data not shown), both
strains show an abundance of the Ams1YFP fusion protein within
accumulated subvacuolar vesicles in cells starved for nitrogen (Fig.
4). Therefore, like prAPI, Ams1 also utilizes autophagy to reach the
vacuole. Based on these data, we propose a model for Ams1 import shown
in Fig. 5.
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Fig. 5.
Model for Ams1 delivery to the vacuole.
Like prAPI, Ams1 assembles into an oligomer in the cytosol and is
sequestered by membrane that forms either a Cvt vesicle (nutrient-rich
conditions) or an autophagosome (starvation conditions). It is not
known whether Ams1 associates with the prAPI-containing Cvt complex.
The completed vesicle targets to and fuses with the vacuole, releasing
the Cvt body or autophagic body into the vacuole lumen. Subsequent
vesicle breakdown releases the cargo. Unlike prAPI, proteolytic
maturation of Ams1 does not occur concomitant with vacuolar import.
Oligomeric Ams1 becomes peripherally associated with the vacuole
membrane.
-mannosidase than the yeast enzyme, as expected.
Extensive sequence comparison between the known
-mannosidases (EC
3.2.1.24) in fungi with a rat homologue has been described (42). None of these protein family members have any classical signal sequence or
membrane-spanning domains, suggesting that they may not transit through
the secretory pathway. Analysis of the known Ams1 homolog from rat
suggested that it is localized to the ER and the cytosol (43, 44);
however, to our knowledge, the possibility of a lysosomal localization
for this protein has not been fully explored. Ams1 and its homologs
were recently categorized as the most ancestral
-mannosidase family
in a global comparison within the
-mannosidase superfamily (45).
Once the Ams1-like human homolog has been identified, it will be
interesting to observe its intracellular localization and cell
type/developmental expression pattern and determine whether a Cvt-like
cytoplasm to lysosome pathway exists in addition to the established
autophagy pathway in humans (46). Perhaps the ancestral targeting
pathway has been maintained for this putative protein. Continued study
of the cytoplasm to vacuole targeting pathway in yeast and a search for
additional Cvt cargo will broaden our understanding of vacuole/lysosome biogenesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. John Kim, Sidney Scott, and
Sarah Teter for critically reading the manuscript; Dr. Tanya Harding
for constructing the ams1 strain; and Drs. Neta Dean,
Dennis Thiele, Michael Kuranda, and Phillips Robbins for their generous
gift of plasmids.
![]() |
FOOTNOTES |
---|
* This work was supported by Public Health Service Grant GM53396 from the National Institutes of Health (to D. J. K.) and National Science Foundation Plant Cell Biology Training Grant DIR 90-14274 (to M. U. H.).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: Dept. of
Biology, University of Michigan, 830 N. University Ave., Ann Arbor, MI 48109. Tel.: 734-615-6556; Fax: 734-647-0884; E-mail:
klionsky@umich.edu.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M101150200
2 Scott, S. V., Guan, M. U., Kim, J., and Klionsky, D. J. (2001) Molec. Cell, in press.
3 Andrei-Selmer, C., Knüppel, A., Satyanarayana, C., Heese, C., and Schu, P. V. (2001) J. Biol. Chem. 276, 11606-11614.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ER, endoplasmic
reticulum;
Ams1, vacuolar -mannosidase;
Apg, autophagy;
API, aminopeptidase I;
CPY, carboxypeptidase Y;
Cvt, cytoplasm to vacuole
targeting;
FM 4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium
dibromide;
HA, hemagglutinin epitope;
prAPI, precursor aminopeptidase
I;
SD
N, synthetic minimal medium lacking nitrogen;
SGd, synthetic
minimal medium containing 3% glycerol;
SMD, synthetic minimal medium
containing nitrogen;
YFP, yellow fluorescent protein;
YNB, yeast
nitrogen base;
OG, n-octyl-
-D-glucopyranoside;
MES, 2-(N-morpholino)ethanesulfonic acid;
MOPS, 3-(N-morpholino)propanesulfonic acid;
kb, kilobase pair(s);
PAGE, polyacrylamide gel electrophoresis;
TSB, Tris salts buffer.
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