Section of Microbiology, University of California, Davis, California 95616
Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.
The faithful transport of resident organellar proteins
is a hallmark in maintaining the functional characteristics and integrity of eukaryotic cells. All nuclearencoded proteins delivered to their respective organelles
must cross at least one membrane barrier. Posttranslational delivery of proteins into membrane compartments occurs by three known mechanisms: a protein-conducting
channel, as observed for example in the import of proteins
into mitochondria and ER (Pfanner and Neupert, 1990 Proteins arrive at their respective organelles in either a
partially unfolded state or a fully folded conformation.
Most proteins enter the ER cotranslationally, while those
that cross the membrane after synthesis assume an extended conformation to pass through the translocation
complex in the ER membrane (Rapoport et al., 1996 A vacuolar version of a channel equivalent to the nuclear pore complex would allow the passage of folded proteins into the vacuole lumen. However, there is no morphological evidence for such pore complexes on the vacuole
membrane. Furthermore, the vacuole maintains a membrane potential through the action of a vacuolar ATPase
(Klionsky et al., 1990 In contrast to the extended conformation used by proteins imported into mitochondria and ER, conformational
studies of peroxisomal proteins revealed that substrates
destined for this organelle can enter the peroxisomal matrix in a fully folded state. In fact, large, preassembled oligomeric complexes can be imported into the peroxisomal
lumen (Subramani, 1993 The majority of resident vacuolar proteases are transported posttranslationally via part of the secretory pathway. Upon vacuolar delivery, most of these hydrolases are
processed by cleavage of a propeptide region to produce
the active mature form of the enzymes. In contrast, aminopeptidase I (API)1 is delivered to the vacuole by the
nonclassical cytoplasm to vacuole targeting (Cvt) pathway
(Klionsky et al., 1992 The study of the mechanism of API import promises to
contribute to our understanding of the basic processes of
not only the Cvt pathway but also autophagy. Autophagy
plays a central role in protein and organelle turnover in all
eukaryotic cells by delivering cytoplasmic components to
the lysosome/vacuole. In addition, autophagy has been implicated in cellular remodeling during development and
differentiation and removal of damaged cellular components, and is critical for survival during stress conditions
such as nutrient deprivation (Glaumann et al., 1981 The molecular components and mechanisms of both autophagy and the Cvt pathway remain to be elucidated. In
this study, we examined the oligomerization state of API
during its import process to understand how it enters the
vacuole. Previous studies have indicated that API exists in
the vacuole as a dodecamer of identical subunits (Metz et al.,
1977 Strains and Media
The Saccharomyces cerevisiae strains used in this study were SEY6210,
MAT cvt strains 1 to 17 were derived from SEY6210 and SEY6211 (Harding
et al., 1995 Reagents
The Vistra enhanced chemifluorescence (ECF) Western Blotting System
was obtained from Amersham Corp. (Arlington Heights, IL); oxalyticase
was from Enzogenetics (Corvallis, OR); Express35S35S-label was from Dupont/NEN Research Products (Boston, MA); proteinase K, Pefabloc, and
the molecular mass standards used for the native gel and glycerol gradient
analyses were obtained from Boehringer Mannheim Corp. (Indianapolis,
IN); Sulfo-NHS-Biotin and avidin agarose were from Pierce (Rockford,
IL); all other reagents were from Sigma Chemical Co. (St. Louis, MO).
Antisera against the following proteins were prepared as described previously: API (Klionsky et al., 1992 Preparation of Crude Cell Extracts, Spheroplasting,
Cell Fractionation, and Labeling
Labeling of whole cells has been described previously. In brief, cells were
grown to an OD600 of 1 and resuspended in synthetic minimal medium
(SMD; 0.067% yeast nitrogen base, 2% glucose, and auxotrophic amino acids and vitamins as needed) at 20-30 OD600/ml. The resuspended cells
were labeled with 10-20 µCi of 35S Express label/OD600 for the indicated
times and temperatures, followed by a chase reaction in which the labeled
cells were diluted to 1 OD/ml in SMD supplemented with 0.2% yeast extract, 4 mM methionine, and 2 mM cysteine.
For preparation of crude cell extracts used for glycerol gradient analysis, 5-15 OD600 units of labeled or unlabeled cells were resuspended in 300 µl
of 20 mM K-Pipes, pH 6.8, containing a cocktail of protease inhibitors (10 mM benzamidine, 4 mM Pefabloc or 2 mM PMSF, 2 µg/ml pepstatin, and
10 mM NaN3). Acid-washed glass beads were then added to a level 50%
of the resuspended cell volume. The cell suspension was lysed by vortexing for 1 min, and the cell extract was collected after a 3-min centrifugation at 12,000 g.
Spheroplasts were prepared using a modified procedure of one previously described (Klionsky et al., 1992 Subcellular fractionations of spheroplasts by differential osmotic lysis
has been described previously (Scott and Klionsky, 1995 Protease Studies
Spheroplasts of the THY101 (ape1 Biotinylation Studies
Spheroplasts were subjected to pulse/chase analysis and subcellular fractionation exactly as in the protease protection experiment above. The pellet fraction (from 5 OD600 of radiolabeled spheroplasts) was resuspended
in 500 µl of import buffer (pH adjusted to 7.4) containing 0.5 mg/ml SulfoNHS-Biotin in the presence or absence of 0.2% Triton X-100. The samples were incubated for 20 min at room temperature. The reaction was
quenched by the addition of 100 mM Tris-Cl, pH 7.5, followed by precipitation with 10% trichloroacetic acid. Proteins were recovered by immunoprecipitation with antibodies against API and CPY followed by recovery
on avidin agarose beads (Scott and Klionsky, 1995 Glycerol Gradients, Immunoprecipitations, and
Western Blotting
For gradient analysis, 200 µl of cell extracts or fractionated spheroplast
samples were loaded on a 1.8-ml, 20-50% glycerol step-gradient prepared
in 20 mM K-Pipes, pH 6.8, with a cocktail of protease inhibitors. The gradients were spun in a centrifuge (model TL-100; Beckman Instrs., Fullerton, CA) for 4 h at 270,000 g, and at 15°C using a TLS-55 rotor (Beckman
Instrs.). Fractions were collected from the top of the gradients and immunoprecipitated as previously described (Klionsky et al., 1992 Quantitation of immunoprecipitations and Western blots was performed using a phosphorimager (model Storm; Molecular Dynamics, Sunnyvale, CA) equipped with both phosphorescent and chemifluorescent scanning capabilities.
Native Gel Sample Preparation and Electrophoresis
Sample preparation, native gel electrophoresis, and Hedrick-Smith analysis procedures have been previously described in detail (Hedrick and
Smith, 1968 Precursor and Mature API Are Both Dodecamers in
Steady-State Conditions
The native state of API was examined by glycerol density
gradients and native gel electrophoresis. Cell extracts were
prepared from wild-type cells and separated on 20-50%
glycerol density gradients as described in Materials and
Methods. After centrifugation, the proteins in the collected fractions were precipitated and subjected to Western blot analysis. Under steady-state conditions, the majority of API in wild-type cells exists as the processed, mature form of the molecule. However, when cells were
grown to midlog phase, a small population of precursor
API could also be detected (Fig. 1 A). The peak concentrations of both precursor and mature API appeared in
fraction 7 of the gradient (Fig. 1 A), cofractionating with
the 669-kD molecular mass standard, thyroglobulin. This
finding is consistent with the dodecameric stoichiometry of mature API subunits (12 × 50 kD = 600 kD) that was
proposed previously (Metz et al., 1977
To obtain a separate estimation of the molecular mass
of the API oligomer, native gels were run and analyzed by
the Hedrick-Smith method (Hedrick and Smith, 1968 Oligomerization of Precursor API Is an Early Step in
Its Import
The half-time of API import and processing is 30-45 min
(Klionsky et al., 1992 After a 2-min pulse without a chase, the majority of labeled precursor API was recovered in fractions 2 and 3, consistent with the size of the precursor API monomer (61 kD;
Fig. 2, top panel). Interestingly, even at this early time
point, a small peak at fraction 7 corresponding to oligomeric precursor could be detected. After a 3-min chase,
the relative distribution of precursor API monomer and
oligomer was reversed, with the majority of the labeled
precursor API assembling into the oligomeric form and
the concomitant depletion of the labeled monomer (Fig. 2, second panel). Oligomerization of labeled precursor API
was nearly complete after 6 min of the chase reaction, and
no monomer was detected after 10 min of chase (Fig. 2,
third and fourth panels, respectively). These data indicate
a half-time of oligomerization of ~2 min. Therefore, the in
vivo kinetics of precursor API oligomerization are far
more rapid than the half-time of API maturation, suggesting that oligomer assembly is an early step in the import of
API into the vacuole.
Precursor API Oligomerizes in the Cytoplasm and Then
Binds to a Membrane
To determine the subcellular location of precursor API
oligomerization, subcellular fractionation experiments were
performed. Cells were converted into spheroplasts, labeled for 5 min, chased for 3 min, and subjected to a differential osmotic lysis procedure that disrupts the plasma
membrane while preserving the integrity of the vacuole
(Scott and Klionsky, 1995
Under these identical labeling and chase conditions, both
the precursor API monomer and dodecamer were detected (Fig. 3 B). After subcellular fractionation, the labeled supernatant and resuspended pellet fractions were
separated by 20-50% glycerol gradients and examined by
immunoprecipitation. Monomeric precursor appears only in the supernatant fraction along with the fully assembled
oligomeric precursor, suggesting that the API assembly
process occurs in the supernatant fraction containing the
released cytoplasm (Fig. 3 B). In contrast, primarily the
oligomeric precursor was detected in the pellet fraction
(Fig. 3 C). Furthermore, oligomeric precursor binding was
very rapid, as 51% of the precursor API dodecamer was
already bound to the membrane fraction after the 3-min
chase reaction. Previous experiments have shown that the
membrane-bound API is on the import pathway as it
chases into the vacuole in vitro (Scott and Klionsky, 1995 cvt and Propeptide Deletion Mutants Are Not Defective
in API Oligomerization
The first amphipathic helix of the API propeptide is critical for proper targeting of the enzyme (Oda et al., 1996
In addition to API propeptide mutants, we identified a
series of chromosomal mutants (cvt) that are defective for
API localization. The cvt mutants were isolated based on
their accumulation of precursor API (Harding et al., 1995 A Unique Temperature-sensitive API
Propeptide Mutant (K12R) Accumulates in the
Membrane Fraction
The oligomerization and membrane-binding steps of API
targeting appear early in the overall import process. We
next examined the oligomeric nature of API during the remainder of the targeting steps. The membrane binding of
precursor API oligomer is followed by import into the vacuole and cleavage of the propeptide by proteinase B (PrB),
which yields mature API (Klionsky et al., 1992
To examine the localization of precursor API in the
K12R mutant at nonpermissive temperature, spheroplasts
were labeled for 10 min at 38°C and chased from 0 to 2 h at
38°C. At each chase time point, samples were separated
into supernatant and pellet fractions after differential osmotic lysis and analyzed by immunoprecipitation. Over
the course of the chase period, the labeled precursor API was depleted from the supernatant fraction and accumulated in the pellet fraction (Fig. 5 A). The kinetics of membrane binding were essentially identical for K12R API at
38°C and the permissive temperature of 30°C (data not
shown). Therefore, the unique targeting defect of the
K12R API mutation did not affect the membrane-binding step, but rather a subsequent step in the import process.
The topology of K12R API accumulated at the membrane-bound stage was examined by protease digestion
experiments. As above, labeled K12R API was allowed to
accumulate in the membrane fraction of spheroplasts by a
10-min labeling and a 30-min chase reaction at 38°C. After
differential osmotic lysis, the supernatant and pellet fractions were treated with proteinase K and examined by immunoprecipitation (Fig. 5 B). The vacuoles were shown to
be intact as the majority of the lumenal vacuolar hydrolase
CPY was localized in the pellet fraction, while the cytosolic
marker, PGK, was concentrated in the supernatant fraction
(Fig. 5 B, right). The majority of K12R API that accumulated in the membrane fraction was accessible to protease
digestion, suggesting that the bound precursor API was
exposed to the cytoplasmic environment and not protected within a lumenal compartment (Fig. 5 B, left). The
small amount of precursor API remaining in the pellet
fraction after protease treatment is consistent with unlysed
spheroplasts that segregated to the pellet fraction (Fig. 5
B, right); this precursor API population was digested upon
addition of detergent (Fig. 5 B, left).
The results of the protease-protection experiment were
confirmed by assessing the accessibility of K12R API and
CPY recovered in the pellet fraction to modification by
Sulfo-NHS-biotin, a water-soluble cross-linker conjugated
to biotin. Proteins modified by this cross-linker can be specifically recovered by precipitation with avidin agarose
beads (Scott and Klionsky, 1995 To determine if the K12R import block was thermally
reversible, temperature shift experiments were performed.
Cells were labeled for 10 min and chased for 30 min at
38°C to accumulate K12R precursor API on the membrane. These cells were then shifted to 30°C for various
lengths of time before immunoprecipitation (Fig. 5 D, top). After 20 min at the permissive temperature, essentially all of the API was still present as the precursor form.
However, between 20-40 min after the 30°C shift (Fig. 5
D, top, two-headed arrow), maturation of API increased
from 10 to 56%. Thus, the thermal reversal of the targeting defect allowed nearly half of the labeled precursor API
to enter the vacuole and become processed to the mature
hydrolase between these two time points. Complete maturation of the precursor API was observed after 90 min at
30°C, whereas labeled cells maintained at 38°C during the
shift phase of the experiment remained defective for import (Fig. 5 D, middle). The thermal reversibility of the
K12R API indicated that it was on the authentic import
pathway.
The K12R ts Mutant Traces the Import of API
Oligomer during the Last, Rate-limiting Stages of the
Cvt Pathway
A key question that we wanted to address was the oligomeric nature of precursor API during the transport step.
The K12R API bound to the membrane at the nonpermissive temperature represented a synchronized population
of labeled precursor, making it possible to follow its import into the vacuole upon reversal of the accumulation phenotype. Cells were labeled for 10 min and chased for
30 min at 38°C to accumulate precursor API oligomers on
the membrane (Fig. 5 A). The labeled cells were then
shifted to 30°C to reverse the block and allow precursor
API to chase into the vacuole. The oligomeric state of API
was examined during the 20-40 min window of the shift
phase, when nearly half of the accumulated precursor API
was chased into the vacuole and processed. Samples were
removed at 20, 27, 34, and 40 min of the 30°C shift period
and analyzed by glycerol gradients and immunoprecipitation. As expected, at the 20-min shift point all of the API
is present as the precursor form, and by 40 min we see
approximately half of the protein as the mature species
(Fig. 6). At all time points, however, we detected only the
oligomeric form of API, either as the precursor or mature dodecamer recovered in fraction 7 of the glycerol gradients. The kinetic examination of import indicated that oligomeric precursor API maintained its dodecameric state
during the period of transport from the membrane-bound
step to entry and processing in the vacuole (Fig. 6).
While we could not detect monomeric API during the
import process, it is possible that the signal for a monomeric translocation intermediate was below our detection
level. However, if such disassembly occurred, then the
API monomers would be exposed to the hydrolases of the
vacuole lumen before reassembly into oligomers. In addition, a protein that goes through a translocation pore
would probably assume a partially unfolded conformation
(Schatz and Dobberstein, 1996 Our study demonstrates that a large oligomeric protein is
capable of being imported into the vacuole in a biosynthetic manner and suggests a possible mechanism of API
transport by the Cvt pathway. Examination of the oligomeric state of API during transport allowed us to dissect
this process into a number of discrete steps. Kinetic analyses indicated that precursor API oligomerizes rapidly into
a homododecamer (Fig. 2) as the majority of precursor
monomers assembled into oligomers within the first 5 min
of the pulse-label/chase reaction. Subcellular fractionation
localized the precursor API assembly process to the cytosol (Fig. 3 B). The subsequent membrane binding of the
oligomeric precursor API was also rapid, with over half
of the labeled precursor associating with the membrane
within this brief labeling and chase period. These results
demonstrate that the early steps of API import, oligomerization and membrane association, are not rate limiting,
and the time required for these early steps constitutes only
a fraction of the time needed for API import and maturation in the vacuole.
Steady-state analyses of the cvt and API propeptide deletion mutants indicated that neither encodes gene products or contains API sequence determinants necessary for
API oligomerization, respectively (Fig. 4). However, a K12R
point mutation in the propeptide resulted in a unique targeting defect that was useful for our examination of the
later stages of API import.
The majority of the time required for API import was
invested in the transport of the enzyme into the vacuole
lumen from the membrane-bound state, indicating that
this stage of the import process is rate limiting. To study
this event, we exploited the novel targeting defect of K12R
API. This temperature-sensitive mutant accumulated the
oligomeric precursor at the membrane-associated step
(Fig. 5 A), with the hydrolase exposed to the cytoplasmic milieu as determined by its accessibility to protease and biotinylated cross-linker (Fig. 5, B and C). Furthermore, the
thermal reversibility of the targeting defect allowed us to
kinetically examine the oligomerization state of API during
the membrane transport event (Figs. 5 D and 6). Between
20-40 min of shift to the permissive temperature, nearly
half of the membrane-accumulated oligomeric precursor
was chased into the vacuole to the mature form (Fig. 5 D,
top). When the oligomeric state of API was examined during this process, no disassembly of oligomeric precursor
API was observed (Fig. 6). These results strongly suggest
that the dodecameric precursor API maintains its oligomeric conformation throughout its import into the vacuole.
Oligomerization and membrane targeting do not appear
to be rate-limiting steps in API import. The efficient assembly of the API dodecamer may require cytosolic chaperones for proper oligomerization as well as to prevent protein aggregation (Hartl, 1996 The target of the newly synthesized oligomeric precursor is a membrane fraction containing intact vacuoles. However, whether the initial membrane binding of precursor
API oligomer occurs at the vacuole surface or perhaps at a
prevacuolar compartment remains to be resolved. Overproduction of precursor API causes the accumulation of
the cytosolic precursor, suggesting that there is a saturable
receptor for API import (Klionsky et al., 1992 Others have suggested that API uses a translocation
mechanism to enter the vacuole (Seguí-Real et al., 1995 The transport of oligomeric API across the vacuolar
membrane is consistent with a vesicle-mediated mechanism.
Vesicle-mediated transport events, including vacuolar vesicle fusion, are dependent on GTP-binding proteins (Zerial and Stenmark, 1993 In the first model, the precursor oligomer binds directly
to the vacuole and enters in an endocytic-like manner (Fig.
7 A). This is followed by the breakdown of the API-containing vesicles and processing of precursor API to the mature hydrolase. Alternatively, oligomeric precursor API
may initially bind to a prevacuolar compartment before
delivery to the vacuole via transport vesicles. The second model (Fig. 7 B) is based on the significant genetic overlap
of the Cvt pathway with autophagy, which suggests that
these two routes to the vacuole share largely the same cellular machinery (Harding et al., 1996
The molecular details of the autophagy and Cvt pathways are not well understood. This study places the events
of API oligomerization and import into discrete steps
along the Cvt pathway. A detailed analysis of API import
will help elucidate the molecular basis of both the Cvt and
autophagy pathways.
;
Hannavy et al., 1993
; Egner et al., 1995
; Rapoport et al.,
1996
); a large nuclear pore-like complex (Davis, 1995
;
Görlich and Mattaj, 1996
); and by vesicle-mediated transport and fusion events (Rothman and Wieland, 1996
). The
conformational state of proteins during this translocation
step limits and defines the possible mechanisms by which
proteins can be delivered to their final destination.
).
Most mitochondrial proteins enter the organelle posttranslationally and must assume a partially unfolded state
before translocation across the membrane through a proteinaceous channel (Hannavy et al., 1993
; Hachiya et al.,
1995
; Ryan and Jensen, 1995
). Small monomeric proteins
or peptides may also be translocated across membranes
through ATP binding cassette transporters (Egner et al.,
1995
). No proteins are known to enter the vacuole cotranslationally, and a protein-conducting channel has not been
identified in the vacuole membrane.
). The corresponding requirement for
a sealed membrane would preclude such pore complexes
from existing on the vacuole.
; Rachubinski and Subramani, 1995
). The dimeric thiolase, CAT trimers, and alcohol oxidase octamers all serve as substrates for import into the
peroxisome (Walton et al., 1992
; Glover et al., 1994
; McNew and Goodman, 1994
). The lack of ideal marker proteins that undergo proteolytic maturation steps, however,
has complicated the analysis of this pathway. Accordingly,
whether peroxisomal substrates enter the organelle by a
translocation pore or through a vesicular mechanism remains to be resolved.
; Harding et al., 1995
). After synthesis as a cytosolic 61-kD precursor, API becomes proteolytically processed in the vacuole by the removal of its NH2-terminal propeptide, resulting in the 50-kD mature protease
(Klionsky et al., 1992
). Thus, the molecular mass shift of
API upon vacuolar delivery serves as a useful marker for
correct import. Using this criterion, a set of mutants (cvt)
defective in API processing was recently isolated (Harding
et al., 1995
, 1996). Surprisingly, the cvt mutants show extensive genetic and phenotypic overlap with two sets of
mutants defective in autophagy, apg and aut (Tsukada and
Ohsumi, 1993
; Thumm et al., 1994
), which suggests that
the Cvt pathway and the autophagy pathway use much of
the same cellular machinery (Harding et al., 1996
; Scott et al., 1996
).
; Marzella and Glaumann, 1987
; Mortimore et al., 1989
; Hilt and
Wolf, 1992
; Egner et al., 1993
; Dunn, 1994
). Despite the
genetic overlap between the autophagy and cvt mutants,
some distinctions between the pathways are still apparent.
Whereas bulk autophagy is a nonselective, degradative pathway, the delivery of API is a selective, biosynthetic
process. Similarly, vacuolar protein uptake by bulk autophagy is relatively slow and cannot account for the rapid
kinetics of API import (Egner et al., 1993
; Knop et al.,
1993
; Scott et al., 1996
). Finally, import of API is constitutive, occurring under vegetative growth as well as starvation conditions.
; Löffler and Röhm, 1979
). We hypothesized that examining the kinetics of API assembly and transit through the Cvt pathway would offer insight into the question of
whether large oligomeric complexes are imported into the
vacuole as well as elucidating possible intermediate steps
of the biosynthetic Cvt pathway. Further, we used a unique
API-targeting mutant that allowed us to kinetically examine
the assembly state of API during the membrane crossing
event. Our findings indicate that precursor API is rapidly
oligomerized into a dodecameric complex that is subsequently transported into the vacuole without disassembly.
Materials and Methods
leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801 suc2-
9 GAL and
SEY6211, MATa leu2-3,112 ura3-52 his3-
200 trp1-
901 ade2-101 suc2-
9 GAL (Robinson et al., 1988
); DYY101 MAT
leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801 suc2-
9 GAL ape1
::LEU2 (Klionsky et al.,
1992
); THY101 MAT
leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801
suc2-
9 GAL ape1
::LEU2 (Oda et al., 1996
).
, 1996). The plasmid construction of the API propeptide deletions (
3-5,
6-8,
9-11,
12-14,
15-17,
18-20,
25-27,
28-30,
31-
33,
34-36,
37-39,
40-42,
2-45) and the K12R API mutation were described previously (Oda et al., 1996
). The propeptide deletion mutation
plasmids were introduced into strain DYY101, while the K12R mutant
was transformed into strain THY101.
), carboxypeptidase Y (CPY) and proteinase A (PrA) (Klionsky et al., 1988
), and alkaline phosphatase (ALP) (Klionsky and Emr, 1989
); antiserum against phosphoglycerate kinase (PGK) was provided by Dr. Jeremy Thorner (University of California, Berkeley, CA) (Baum et al., 1978
).
). Cells were grown in SMD to an
OD600 of 1 and incubated for 15 min at 30°C in a buffer of 0.1 M Tris-SO4,
pH 9.4, 10 mM DTT. After a 5,000-g spin for 5 min, the cells were resuspended in an osmotically supportive spheroplast labeling medium (1 M
sorbitol, 1% glucose, 1% proline, Wickerham's salts, pH 7.5 [Guthrie and
Fink, 1991
]), containing 1 µg/OD600 of oxalyticase and incubated for 30 min at 30°C. After a 5-min spin at 3,000 g, the spheroplasts were resuspended in fresh spheroplast labeling medium, pH 5.0, at 20-50 OD600/ml
and labeled with 10-20 µCi of 35S Express label/OD600 for 5-10 min at the
indicated temperatures. The labeling reaction was chased by diluting the
spheroplasts to 1-3 OD600/ml in SMD supplemented with 0.2% yeast extract, 1 M sorbitol, 4 mM methionine, and 2 mM cysteine for the times and temperatures indicated.
). The pellet fraction was resuspended in 20 mM K-Pipes, pH 6.8, with protease inhibitor
cocktail to release the vacuolar lumenal contents as well as to release API
bound to the pellet.
) strain harboring the K12R API single copy plasmid were labeled for 5 min, followed by a chase reaction for
30 min at 38°C. The labeled spheroplasts were subjected to differential osmotic lysis in import buffer (20 mM K-Pipes, pH 6.8, 100 mM sorbitol, 100 mM KCl, 50 mM KOAc, 5 mM Mg[OAc]2). Permeabilized cells were then
fractionated by centrifugation at 5,000 g for 3 min, resulting in a supernatant fraction and a pellet fraction that was then resuspended in import
buffer. The pellet fraction contains intact vacuoles (Scott and Klionsky, 1995
). The fractions were treated with 100 µg/ml proteinase K alone, or
with the addition of 0.2% Triton X-100 for 15 min, on ice. Nonradioactive
pellet and supernatant fractions were added to the labeled supernatant
and pellet samples, respectively, to keep the protein concentration constant during the protease digestion procedure and subsequent protein recovery steps.
). Nonradioactive pellet
and supernatant fractions were added to the labeled supernatant and pellet samples, respectively, to keep the protein concentration constant during the cross-linking and protein recovery procedures.
). The Western
blotting procedure described previously (Oda et al., 1996
) was modified and immunodetection was executed using a Vistra ECF chemifluorescent substrate (Amersham Corp.). Identical gradients were run with a mixture
of molecular mass standards consisting of ovalbumin (45 kD), aldolase
(158 kD), catalase (240 kD), apoferritin (450 kD), and thyroglobulin (669 kD); the collected fractions were precipitated as described above and subjected to SDS-PAGE and Coomassie brilliant blue staining.
; Tomashek et al., 1996
).
Results
; Löffler and Röhm,
1979
). In addition, the appearance of precursor API in
fraction 7 suggests that it also forms a dodecameric complex (12 × 61 kD = 732 kD). To confirm that these large
complexes were in fact API homooligomers, cross-linking studies were performed using radiolabeled cell extracts.
As before, precursor API-containing cross-linked products were recovered from fraction 7 of glycerol gradients
(data not shown). On reduction of the cross-link, only API
was detected, suggesting that the API oligomer recovered
from fraction 7 is a homooligomer. These data do not rule
out the possibility, however, that other proteins transiently
associate with the API oligomer.
Fig. 1.
Molecular mass determination of native precursor and
mature API under steady-state conditions. (A) Glycerol gradient
analysis of precursor and mature API under steady-state conditions. Wild-type (SEY6210) cells were grown to midlog phase,
lysed with glass beads, and the resulting cell extracts were separated on 20-50% glycerol gradients. Collected fractions were subjected to Western blotting with antiserum to API. The reaction of
a chemifluorescent substrate (ECF) with an alkaline phosphatase-
conjugated secondary antibody allowed for the quantitation of
the Western blots by a chemifluorescence scanner (Molecular
Dynamics) as shown in the graph corresponding to the Western
blot signals. Molecular mass standards indicated are hen egg albumin (45 kD), aldolase (158 kD), catalase (240 kD), ferritin (450 kD),
and thyroglobulin (669 kD). Both mature (mAPI) and precursor
API (prAPI) peaks appeared in fraction 7, cofractionating with
the thyroglobulin molecular mass standard. (B) Hedrick-Smith
calculation of the molecular masses of precursor and mature API.
Relative mobilities were measured for protein standards resolved
on native gels of 4.0 to 5.5% acrylamide and the negative slopes
were determined by plotting the relative mobilities as a function
of gel percentage. A standard curve was then generated by plotting the negative slopes of the protein standards as a function of
molecular mass. Extracts from wild-type and pep4 strains were
run on the same gels, and their molecular masses were calculated
with reference to the standard curve. Molecular masses of mAPI
and precursor API were determined to be 592 and 752 kD, respectively.
[View Larger Version of this Image (28K GIF file)]
; Tomashek et al., 1996
). Native gels ranging from 4 to 5.5%
acrylamide were run on extracts from wild-type and pep4
strains, and the relative mobilities of both mature and precursor API were measured. Molecular mass standards were
run on the same gels, blotted, and stained with Amido black as cited in Materials and Methods. The relative mobilities
were plotted as a function of gel percent and the negative
slopes of these graphs were then plotted as a function of
molecular mass (Fig. 1 B). The molecular masses of mature API from a wild-type strain and precursor API from a
pep4
strain were determined to be 592 and 752 kD, respectively, consistent with the predicted molecular mass of
600 kD for the mature API dodecamer and 732 kD for the
dodecameric precursor hydrolase (Fig. 1 B). Because the
native gel electrophoresis analyses were in close agreement with the glycerol gradient results, subsequent kinetic
studies were performed using the gradient method.
). We wanted to determine the point
during biosynthesis at which API oligomerization occurs.
To observe the oligomerization process in vivo, whole cells
were labeled for 2 min, followed by chase reactions of 0, 3, 6, and 10 min with excess cold methionine and cysteine.
After each chase point, the labeled cells were immediately lysed with glass beads and centrifuged for 1 min to remove
cellular debris, and the extracts were separated on 20-50%
glycerol density gradients. Fractions were collected from
the gradient and analyzed by immunoprecipitation. Oligomerization did not appreciably occur after cell lysis (data not
shown), presumably because of the resulting substantial
dilution.
Fig. 2.
Oligomerization kinetics of precursor API. Wild-type
cells were pulse labeled for 2 min at 30°C followed by nonradioactive chase reactions. Aliquots were removed at the indicated
chase times and lysed with glass beads, and the resulting cell extracts were separated on 20-50% glycerol gradients. Fractions
were collected, immunoprecipitated with antiserum to API, and
resolved by SDS-PAGE. Molecular mass standards corresponding to the fractions: 45 kD, fraction 2; 158 kD, fraction 4; 240 kD,
fraction 5; 450 kD, fraction 6; and 669 kD, fraction 7. Quantitation of the radioactive signals was performed using a phosphorimager (model Storm; Molecular Dynamics).
[View Larger Version of this Image (23K GIF file)]
). Subsequent centrifugation resulted in a cytosolic supernatant fraction and an organellar membrane fraction that included the unlysed vacuoles.
The faithful segregation of vacuolar markers (ALP and
PrA, vacuolar membrane and lumenal hydrolases, respectively) to the pellet fraction and a cytosolic marker (PGK)
to the supernatant fraction indicated the efficiency of this
subcellular fractionation procedure (Fig. 3 A).
Fig. 3.
Oligomeric precursor API assembly occurs in the cytoplasm before membrane binding. Spheroplasts were labeled for 5 min at 30°C followed by a 3-min nonradioactive chase reaction.
The samples were subjected to differential osmotic lysis and separated into a supernatant fraction and a pellet fraction containing
intact vacuoles. An aliquot of the supernatant and pellet fractions
was removed and immunoprecipitated with the indicated antisera, and the remainder was separated on 20-50% glycerol gradients. (A) Quantitation of the pellet fraction immunoprecipitated
with vacuolar markers PrA and ALP, and the cytosolic marker
PGK. The recovery of marker protein in the pellet was quantified
using a Storm phosphorimager. The percent recovery was calculated as the ratio of the protein in the pellet fraction to the protein in the pellet and supernatant fractions. The supernatant
(sup) fraction (B) and the pellet fraction (C) were separated on a
glycerol gradient and immunoprecipitated with antiserum to
API. Quantitation of the radioactive signals, represented by the
graphs, indicates that the supernatant fraction contains both the
precursor monomer and oligomer, while only the precursor API
oligomer is bound to the pellet fraction.
[View Larger Version of this Image (24K GIF file)]
).
These findings indicate that precursor assembly in the cytoplasm and subsequent membrane binding mark the
early steps of API import to the vacuole.
).
Specifically, deletions in this region inhibit API from binding to the membrane fraction, thus preventing subsequent
import and processing to the mature form (Oda et al.,
1996
). We examined whether the targeting defect in these
propeptide deletion mutants was caused by an oligomerization defect. Cell extracts were prepared from the DYY101
strain deleted for the gene encoding API (ape1
) but harboring a single copy plasmid encoding API with the propeptide deletions. Western blot analysis of glycerol gradient fractions indicated that precursor API in all of the
propeptide deletion mutants peaked in fraction 7 (Fig. 4),
indicating that it was properly oligomerized. In addition, an API molecule with a deletion of the entire propeptide
also formed API oligomers (data not shown). These results indicate that, while the propeptide is necessary for
binding to the membrane fraction and subsequent import
of API into the vacuole, it does not encode any sequence
determinants necessary for API oligomerization. In addition, these results demonstrate that dodecameric precursor API assembly can occur in the absence of membrane
binding or transport to the vacuole, in agreement with the
kinetic and subcellular fractionation data (Figs. 2 and 3).
Fig. 4.
API-targeting mutants are not defective in API oligomerization. Strain DYY101 (ape1) harboring a single copy plasmid encoding the propeptide deletions was grown to midlog
phase, lysed with glass beads, and separated on a 20-50% glycerol gradient. The
9-11 API deletion mutant shown here is a
representative example of the analysis of the cvt (1 to 17) and
propeptide deletion mutants (
3-5,
6-8,
9-11,
12-14,
15-
17,
18-20,
25-27,
28-30,
31-33,
34-36,
37-39,
40-42,
and
2-45). Collected fractions were subjected to Western blotting with antiserum to API and the quantitation of the Western
blots (shown in the graph) was performed as in Fig. 1.
[View Larger Version of this Image (25K GIF file)]
,
1996). To examine if the localization defect in any of these
mutants was due to a failure in API oligomerization, cell
extracts of cvt 1 to 17 were examined by glycerol gradients and Western blotting. Native precursor API from all of the
cvt mutants appeared in fraction 7 in glycerol density gradients, consistent with correct precursor API assembly
(data not shown). These results suggest that cvt mutants
do not have defects in genes whose products are necessary
for API oligomerization.
). To study the oligomeric state of precursor API as it enters the vacuolar lumen from the membrane-bound state, we exploited
the unique characteristics of an API-targeting mutant in
which the twelfth lysine residue in the predicted amphipathic helix of the propeptide was changed to an arginine.
The K12R point mutation rendered API defective for import at nonpermissive temperature, 38°C (Fig. 5 D, middle; Oda et al., 1996
), while wild-type kinetics were observed at
permissive temperature, 30°C (Fig. 5 D, bottom).
Fig. 5.
The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the
pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated
with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase
K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was
also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right).
The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in
the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction
was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20-40-min window
of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.
[View Larger Versions of these Images (30 + 45K GIF file)]
). When the pellet fraction
was treated with Sulfo-NHS-biotin, precursor K12R API
was recovered by avidin agarose regardless of whether detergent was present during the cross-linking reaction (Fig.
5 C). In the same samples, biotinylation of mature CPY
and the small amount of mature API that escaped the temperature block required the vacuoles to be solubilized by
detergent before the addition of cross-linker. Together
with Fig. 5 B, these results indicate that the K12R precursor API that accumulates at 38°C is not in the vacuole,
where it would be protected from both protease digestion and biotinylation. The fact that the mutant precursor is accessible to biotinylated cross-linker, while mature vacuolar
hydrolases are not, suggests that it accumulates in a membrane-bound state that is exposed to the cytosolic environment before vacuolar delivery.
Fig. 6.
Membrane-associated API is imported into the vacuole
as an oligomer. The K12R API mutant was pulse labeled for 10 min and chased for 30 min at 38°C and then shifted to 30°C. Samples were removed at the indicated times during the 20-40-min
window of the 30°C shift period and lysed with glass beads, and
the resulting extract was separated by 20-50% glycerol gradients.
Fractions were collected, immunoprecipitated with antiserum to
API, and resolved by SDS-PAGE. Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating
with the thyroglobulin molecular mass standard (669 kD).
[View Larger Version of this Image (63K GIF file)]
). To mimic this disassembled state, we examined the protease susceptibility of newly
synthesized monomeric precursor API. Cells were labeled
for 2 min to yield predominantly monomeric API, lysed with glass beads, and subjected to proteolysis. These experiments indicated that monomeric API was completely
degraded even after a brief treatment with proteinase K
(data not shown), suggesting that newly synthesized API
monomers would not survive the hydrolytic environment
of the vacuolar lumen. In contrast, mature API is resistant
to proteinase K digestion (Oda et al., 1996
). This result provides additional support for our model that API is
maintained as a dodecamer throughout its transport to the
vacuole.
Discussion
; Jaenicke, 1996
). We are
currently pursuing detailed cross-linking studies to isolate
possible transient API-associated cytosolic factors. Although
oligomerization appears to stabilize API from proteolytic degradation (data not shown), a question that remains is
whether oligomerization itself is necessary for correct targeting of the protein or for its enzymatic activity. Preliminary results suggest that the COOH terminus of API may
contain an oligomerization domain; truncations in this region render API incompetent for import and subsequent
processing in the vacuole (Oda et al., 1996
). Although these deletion mutants are also defective for oligomerization (data not shown), the possibility that they are simply
misfolded cannot be ruled out.
). Assembly
of a multimeric receptor may be necessary to accommodate multimeric propeptide binding. Recruitment of additional cytosolic factors may also contribute to the long
half-time of API import.
).
In this model, precursor API binds to the membrane as a
monomer and inserts its propeptide extension into the vacuolar membrane before membrane translocation. Our current findings indicate that precursor API binds to the membrane as a fully assembled dodecamer rather than as a
monomer. In order for membrane-bound API dodecamer
to enter the vacuole through a protein channel, a disassembly of the oligomeric complex into partially unfolded
monomers would be necessary before translocation into
the vacuole. However, examination of the oligomeric state of
API during membrane transport indicates that the dodecameric conformation is maintained throughout the import process. The precursor is directly processed to the mature hydrolase without the appearance of a kinetic intermediate
API form as proposed by others (Seguí-Real et al., 1995
).
The sheer size of the membrane-bound oligomeric precursor and the instability of monomeric API make unlikely its
passage into the vacuole by known posttranslational, translocation mechanisms.
; Nuoffer and Balch, 1994
; Stack
et al., 1995
). The finding that in vitro import of API is inhibited by nonhydrolyzable GTP analogues suggests a role
for GTPases and the potential participation of vesicle intermediates in the Cvt pathway (Scott and Klionsky, 1995
).
We are currently characterizing vesicular intermediates
that mediate API transport (Scott, S.V., and D.J. Klionsky, manuscript in preparation). From our results, two
models of the API import process can be envisioned.
; Scott et al., 1996
).
Specifically, the double-membrane autophagic vesicles (autophagosomes) that nonselectively capture and deliver cytoplasmic contents (Baba et al., 1994
) to the vacuole may
also be used by precursor API for its transport to this organelle. However, unlike autophagy, the delivery of API is
constitutive. Thus, a continuous, basal-level supply of autophagic vesicles would be required for the selective delivery of API to the vacuole. In this model (Fig. 7 B), API
initially binds to putative cusp-shaped progenitors of the
double-membrane autophagosomes (Baba et al., 1994
).
Upon enclosure and autophagosome formation, API is
transported to the vacuole, where the outer vesicle membrane fuses with the vacuole, releasing a single-membrane vesicle into the vacuolar lumen. These vesicles (autophagic
bodies) are then degraded in a PrB-dependent manner
(Takeshige et al., 1992
), exposing the oligomeric precursor
API to the vacuolar lumen for processing.
Fig. 7.
Models for API import via the Cvt pathway. Precursor
API is synthesized and assembled into dodecamers in the cytoplasm, followed by membrane binding. During the rate-limiting
step of the pathway, both models A and B propose a vesicle-mediated mechanism of API entry into the vacuole followed by the
breakdown of the API-containing vesicles and cleavage of the
API propeptide in a PrB-dependent manner to yield the mature
hydrolase. The role of molecular chaperones and the location of
the initial binding of precursor API remain to be resolved. (A)
Oligomeric precursor API directly binds to the vacuolar or prevacuolar membrane before the vesicle-mediated entry into the
vacuole. (B) Genetic analyses have revealed a large overlap between the autophagy and Cvt pathways. In this model, API is delivered to the vacuole via double-membrane autophagic vesicles.
Upon reaching the vacuole, these vesicles fuse to the vacuolar
membrane, releasing a single-membrane vesicle (autophagic body) containing precursor API. This is followed by the breakdown of the vesicles and subsequent processing of the hydrolase
to the mature enzyme.
[View Larger Version of this Image (26K GIF file)]
Received for publication 19 December 1996 and in revised form 24 February 1997.
1. Abbreviations used in this paper: ALP, alkaline phosphatase; API, aminopeptidase I; CPY, carboxypeptidase Y; Cvt, cytoplasm to vacuole targeting; ECF, enhanced chemifluorescence; PGK, phosphoglycerate kinase; PrA and PrB, proteinase A and B; SMD, synthetic minimal medium containing 2% glucose, essential amino acids and ammonium sulfate.We thank K.A. Morano for his advice and assistance and M.U. Hutchins for critical reading of the manuscript.
This work was supported by a National Institutes of Health Molecular and Cellular Biology Training Grant to J. Kim and by a Public Health Service Grant GM53396 from the National Institutes of Health to D.J. Klionsky.