From the Section of Microbiology, University of California, Davis, California 95616
Fungal cells spend most of their time in
stationary phase (1, 2), yet most research has focused on cells in
logarithmic conditions. In the wild, these cells frequently encounter
periods of limiting nutrient conditions. Accordingly, recycling, as
opposed to simply degradation, of macromolecules is a routine and
critical feature of cell biology. It is important to explore the ways
in which cells adapt to environmental changes, including starvation, to
understand cellular physiology. The basic processes of intracellular transport, as well as many of the specific components, are conserved among yeast and higher eukaryotes, making studies in this
experimentally tractable organism relevant to other systems.
The vacuole is the most prominent organelle in a yeast cell, and
it plays a central role in cellular physiology (3). The vacuole is
involved in cytosolic ion and pH homeostasis and is the main storage
site for calcium and other divalent cations. Metabolites such as basic
amino acids and polyphosphate, as well as toxic substances, are also
stored within this organelle. In addition, the vacuole is important in
osmoregulation, sporulation, and regulatory processes that require
degradation. The best known role for this organelle is in protein
turnover; the vacuole has a large number of membrane-bound and soluble
hydrolases (4).
With regard to the topic of protein targeting, the vacuole is one of
the most complex organelles in a eukaryotic cell. Numerous pathways are
used to target both resident hydrolases and substrates destined for
degradation to the vacuole (5, 6). The best characterized route for
delivery of hydrolases to the vacuole is the secretory pathway. Nascent
proteins transit from the endoplasmic reticulum to the Golgi complex,
and following sorting events in the trans-Golgi network, the proteins
are delivered to the vacuole through an endosomal intermediate (7, 8).
A subset of proteins that transit to the vacuole via the secretory
pathway, including alkaline phosphatase
(ALP)1 and Vam3p, use a
partially alternate route, which bypasses the endosome or prevacuolar
compartment (9-12).
Plasma membrane and cell surface proteins targeted for degradation are
delivered to the vacuole by endocytosis (13). Several other degradative
pathways also exist. Macroautophagy is used for the turnover of bulk
cytosol during starvation (14, 15). The vacuolar import and degradation
pathway targets at least the gluconeogenic enzyme fructose
1,6-bisphosphatase into the vacuole under conditions where its enzyme
activity is no longer needed (5, 16). Similarly, peroxisomes (17, 18)
and mitochondria are selectively imported into the vacuole when
environmental signals trigger a cascade leading to their degradation
(5).
Mammalian cells also utilize both micro- and macroautophagic processes
for the delivery of substrates to the lysosome (19-21). A specific
process of lysosomal uptake has been described by Dice and colleagues
(22), who have identified a lysosomal surface receptor that is involved
in recognition of proteins bearing a pentapeptide (KFERQ) motif. This
import mechanism requires members of the hsp70 family including an
intralysosomal hsc73 (23). These data suggest the presence of protein
translocation machinery in the lysosomal membrane, a property not
generally ascribed to this organelle.
In addition to the pathways mentioned above, other routes are used
for protein delivery to the vacuole. One of the recently characterized
mechanisms is the cytoplasm to vacuole targeting pathway used to
deliver the resident hydrolase aminopeptidase I (API) to the vacuole
(24). An examination of the biosynthesis of API revealed differences
from the standard pattern seen with vacuolar proteins that transit
through the secretory pathway. All of the characterized vacuolar
hydrolases that transit through the ER and Golgi complex undergo
proteolytic and/or glycosyl modifications during transit. Following the
removal of cleavable signal sequences, these proteins receive a core
glycosylation. Subsequent carbohydrate additions, upon delivery to and
passage through the Golgi complex, are detected as an increase in
molecular mass. Delivery to the vacuole is often accompanied by the
removal of a propeptide segment. The half-time for vacuolar delivery
through this route is 5-10 min for most of these proteins.
In contrast to the secretory pathway proteins, API is not glycosylated
even though it has N-linked glycosylation sites (24). It
lacks a signal sequence or consensus signal sequence cleavage site (25,
26). In accordance with the lack of a means to enter the secretory
pathway, the precursor form of API is not found within the endoplasmic
reticulum or Golgi complex but rather within the cytosol (24). API
contains a propeptide at the N terminus that is removed in the vacuole
by a proteinase B-dependent reaction. The half-time for
propeptide processing, and presumably vacuolar delivery, is 30-40 min,
substantially longer than that for proteins that utilize the secretory
pathway. Similarly, the import of API is relatively insensitive to
sec mutants, which were isolated based on defects in transit
through the secretory pathway. In particular, Sec gene products that
are specific to the early steps of the secretory pathway, ER to Golgi
complex transport, are not required for API delivery to the vacuole
(24). Many of the vps mutants that were isolated based on
defects in vacuolar targeting of carboxypeptidase Y show essentially
normal processing kinetics for API. Finally, unlike vacuolar proteins
that traverse the secretory pathway, overexpression of API does not
lead to secretion from the cell. These results indicate that API does
not enter the vacuole through the secretory pathway but rather uses an
alternate mechanism to attain its correct subcellular localization.
Many vacuolar hydrolases are synthesized as zymogens containing a
propeptide segment that keeps the enzyme inactive. The maintenance of a
latent state may serve to protect the cell from the hydrolytic activity
prior to the arrival of the enzyme in the vacuole. The propeptides of
vacuolar hydrolases may also be involved in folding and/or targeting of
the hydrolase (4). The propeptides of proteinase A and carboxypeptidase
Y have been shown to contain vacuolar targeting determinants; however,
no consensus sequences have been defined for yeast vacuolar proteins.
The API propeptide is composed of two A series of random mutants was also generated within the API propeptide
(28). All mutations that caused precursor accumulation mapped within
the first helix. The precursor API was shown to reside in the cytosol
in a protease-sensitive form. This result confirmed that the propeptide
mutations caused a defect in targeting and not simply processing of
API. Hence, the first helix of the API propeptide contains information
that is necessary for delivery to the vacuole. The targeting
information in the first and second helices is also sufficient for
vacuolar localization; this segment of API can target a passenger
protein to the vacuole. Hybrid proteins containing the N-terminal
helices of API fused to the green fluorescent protein, are localized to
the vacuole.2
The API propeptide presumably interacts with subcellular sorting
components such as a binding protein and/or receptor. To identify these
sorting components, an in vitro system was established to reconstitute API import (30). The import reaction is
temperature-dependent, having an optimum at approximately
30 °C. In addition, import is inhibited both in vitro and
in vivo at 14 °C. Inhibition at 14 °C suggests that
import does not occur through a proteinaceous channel as in the ER,
mitochondria, or chloroplasts; translocation in these organelles can
occur at temperatures as low as 0 °C, as long as protein unfolding
is not limiting. A block in transport at 14 °C suggests a
vesicle-mediated event.
The in vitro import reaction is also
energy-dependent (30). When import was carried out in the
presence of non-hydrolyzable ATP or GTP analogs, or when ATP was
depleted, processing of the API precursor was blocked. Inhibition of
vacuolar ATPase activity with specific inhibitors or using
vma mutants resulted in reduced processing of API. In these
cases, however, the block may have been indirect and reflect decreased
access to the precursor protein (see below).
The vacuolar localization of API was also analyzed through a classical
genetic approach (31, 32). Because the mature form of API is stable in
the vacuole lumen, wild type cells accumulate this form of the protein.
To identify mutants in the import pathway, mutagenized yeast cells were
screened for precursor accumulation. Mutants were obtained that are
termed cvt, for cytoplasm to vacuole targeting defective.
These mutants accumulate the precursor form of API but (for most) do
not affect vacuolar delivery of proteins that transit through the
secretory pathway. In all but two cvt mutants, precursor API
is located in the cytosol, indicating that these mutants are blocked in
targeting and do not deliver the precursor protein to the vacuole.
To determine if these mutants are unique, complementation studies were
carried out between cvt mutants and other mutant strains known to affect vacuolar protein delivery. The complementation studies
revealed that some of the cvt mutants are probably allelic to certain vps mutants. This is expected because some of the
vps mutants display major defects in vacuole morphology, in
some cases lacking a detectable vacuole; these mutants do not present a
proper target for API delivery. In addition, some of the components
needed for API delivery may reach the vacuole through the secretory
pathway.
A more substantial overlap is detected between the cvt
mutants and two groups of mutants that were isolated based on defects in macroautophagy, the apg and aut mutants
(32-35). This overlap is surprising because there are substantial
differences between the Cvt pathway and macroautophagy. For example,
API import is kinetically slower than secretory pathway transit but is
much faster than macroautophagy. Along these lines, macroautophagy also
shows a lower yield of protein uptake. The Cvt pathway is biosynthetic,
delivering a resident hydrolase to the vacuole. Accordingly, API import
occurs during vegetative growth. In contrast, macroautophagy is a
degradative pathway and, while occurring at a basal level in rich
media, is induced under starvation conditions. Finally, in agreement
with their respective roles in cellular physiology, Cvt import is
specific for API and perhaps other hydrolases, whereas macroautophagy
is nonspecific and is used to deliver bulk cytosol to the vacuole.
To understand the molecular basis for the overlap between the Cvt
and macroautophagic pathways, the native state of API was examined
during the import process. The mature hydrolase was reported to be a
dodecamer in the vacuole (36-38). To determine when oligomerization occurs, and specifically if the precursor is a dodecamer, a kinetic analysis was carried out (39). The results indicated that
oligomerization into a dodecamer occurred rapidly, with a half-time of
approximately 3 min. This is much shorter than the half-time of
processing and indicates that oligomerization is not rate-limiting in
the import process and occurs prior to vacuolar delivery.
Oligomerization studies combined with subcellular fractionation showed
that the oligomer forms in the cytosol; monomeric API can only be found in a soluble fraction whereas a low speed pellet fraction contains exclusively the dodecameric form. The transition from a soluble to
sedimentable form may be indicative of binding to a target membrane
and/or the formation of a pelletable complex.
A temperature-sensitive API propeptide mutant was utilized to follow
the import of API into the vacuole following the initial oligomerization event. An alteration of lysine to arginine at position
12 of the propeptide, K12R, results in a temperature-sensitive targeting phenotype (28); at the nonpermissive temperature, precursor
K12R API accumulates in the pelletable oligomeric form. Because the
K12R phenotype is thermally reversible, it can be used to follow the
stages of import subsequent to the initial oligomerization reaction and
binding event. Analysis of the K12R mutant revealed that precursor API
transits to and imports into the vacuole in the dodecameric form (39).
This makes translocation through a proteinaceous channel unlikely
because the oligomeric precursor is approximately 732 kDa in mass. This
result, coupled with the in vitro and genetic studies,
suggests that precursor API enters the vacuole through a
vesicle-mediated process that is similar to macroautophagy.
The process of macroautophagy had been characterized in yeast
primarily through morphological studies and more recently by molecular
and classical genetic techniques (14, 15, 33). Macroautophagy begins
with the formation of an enwrapping membrane that sequesters cytosol.
Upon completion, this membrane forms a double membrane vesicle that is
termed an autophagosome (AP). The origin of the autophagosomal membrane
is not known. The fact that APs appear to be fairly uniform in size
suggests that they may originate from a pre-existing organelle such as
a specialized region of the ER. However, it is not clear that the
necessary targeting components, needed for subsequent delivery to the
vacuole, would reside in the membrane of this organelle. In addition,
inhibition by cycloheximide (14) suggests that at least some aspects of macroautophagy require de novo protein synthesis.
Following targeting to the vacuole, the outer membrane of the AP fuses
with the vacuole membrane. The machinery needed for targeting, docking,
and fusion have not been well characterized but are likely to be
similar to those involved in similar processes in other parts of the
cell (40-43). For example, the vacuolar t-SNARE Vam3p, which is
required for homotypic vacuole fusion (44), is also needed for API
delivery to the vacuole (45). Along these lines, preliminary evidence
indicates a role for the rab protein Ypt7p and the SEC19
gene product, GDP dissociation inhibitor, in API
import.3
Fusion of the APs with the vacuole allows the release of the interior
single membrane vesicle into the vacuole lumen. This vesicle, termed an
autophagic body (AB), is eventually broken down by vacuolar hydrolases,
allowing access to the lumenal contents. Yeast strains deficient in
vacuolar hydrolase activity accumulate ABs within the vacuole lumen
(14). Breakdown of the AB is pH-dependent (46). The
elevated vacuolar pH in vma mutants stabilizes ABs. This may
explain the apparent block in API processing in vma
mutants.
Applying the morphological data on macroautophagy to API import
allows specific predictions about the Cvt pathway. In particular, if
API is imported by a process similar to macroautophagy, precursor API
should reside in the cytosol in a double membrane vesicle analogous to
an autophagosome. Similarly, the precursor protein should accumulate
within single membrane vesicles, equivalent to ABs, in the vacuole
lumen in mutants that are defective in vesicle breakdown. This model
for API import was tested using mutants defective in various stages of
the localization process.
The Vps18 protein is part of a complex that is required for transport
to, or fusion with, the vacuole membrane (47). A temperature-sensitive vps18 mutant accumulates precursor API (48). Pulse-chase
studies showed that precursor API in the vps18
temperature-sensitive mutant grown in rich medium went from a
protease-sensitive to a protease-insensitive form, consistent with its
enclosure within a membrane-bound compartment. Subcellular
fractionation studies demonstrated that the precursor was not located
within the vacuole, consistent with its sequestration in cytosolic
vesicles.
The cvt17 mutant accumulates precursor API within the
vacuole. Light microscopy indicates that the cvt17 mutant is
defective in vacuolar vesicle breakdown. Precursor API cofractionates
with vacuoles in the cvt17 mutant. Differential osmotic
lysis conditions, allowing lysis of the vacuole membrane but not
subvacuolar vesicles, revealed that the precursor API could be
fractionated away from vacuolar lumenal proteins (48). Protease
protection studies confirmed that the subvacuolar precursor API is
contained within a membrane-enclosed compartment. Analysis of marker
proteins demonstrated that these subvacuolar compartments, termed Cvt
bodies, were distinct from vacuole membrane vesicles.
These results suggested that precursor API resides within both
cytosolic and vacuolar vesicles. The only way for a subvacuolar vesicle
to be derived from the fusion of a cytosolic vesicle with the vacuole
is for the cytosolic vesicle to be double membraned.
The topology of API during import and the morphology of the
transit vesicles were directly examined using immunoelectron microscopy (48, 49). Precursor API is strikingly absent from the majority of the
cytoplasm. Instead of a random distribution, it clusters into
complexes. What are presumed to be initial complexes are apparently
devoid of membrane or at least of a unit membrane structure. The nature
of these complexes, termed Cvt complexes, is not known. The Cvt
complexes are subsequently surrounded by membrane, resulting in the
formation of double membrane Cvt vesicles. Precursor API was also
detected in vesicles in the vacuole lumen in the cvt17 mutant. These morphological data provide direct confirmation of the
biochemical and genetic data suggesting that API import occurs through
a vesicle-mediated process.
Although the data cited above indicated a substantial mechanistic
overlap between the Cvt and macroautophagic pathways, there are still
differences. To directly compare the two pathways simultaneously, a
macroautophagic marker was analyzed. ALP is a resident vacuolar membrane protein that transits to the vacuole through a portion of the
secretory pathway. ALP is a type II integral membrane protein with a
C-terminal propeptide that faces the vacuole lumen and that is removed
upon vacuolar delivery (50). The N terminus of ALP contains a
transmembrane domain that serves as an internal uncleaved signal
sequence that is needed to gain entry into the ER. Removal of a portion
of the N terminus of ALP including the transmembrane domain generated a
construct, Pho8 Pho8 API import occurs under both vegetative and starvation conditions.
Macroautophagic import cannot account for API import under vegetative
conditions. To examine the nature of the import process under
starvation conditions, a morphological analysis was employed (49). API
import was followed during a shift from vegetative to starvation
conditions. During vegetative growth, precursor API in the form of Cvt
complexes was detected in Cvt vesicles as described above. These
vesicles are approximately 150 nm in diameter and contain an
electron-dense core that appears to be distinct from cytosol. Upon
starvation, the Cvt complexes are seen inside autophagosomes. The ratio
of Cvt vesicles to APs decreased as starvation conditions progressed.
The APs are substantially larger than Cvt vesicles, having a diameter
of 400 to 900 nm. In addition, the APs contain cytosol along with the
Cvt complex.
These data suggest that API is imported into the vacuole by two
processes that substantially overlap and that share many components (Fig. 1). Under vegetative conditions,
import occurs through the Cvt pathway. Starvation signals the need for
the turnover of cytosolic proteins. By some mechanism, Cvt vesicles are
no longer synthesized or are modified to become APs. The signaling
mechanism is not known but may involve kinases such as Tor2p (52).
Because API uptake remains rapid and complete under starvation
conditions, specific machinery must still operate to ensure efficient
uptake during starvation. It is interesting to note that API levels
increase substantially during nitrogen limitation (35). The increase in
API synthesis is accommodated by an increase in the import capacity via
the use of APs. If API is a critical hydrolase during starvation, the
cell ensures an adequate supply of this enzyme by connecting its
biosynthesis to macroautophagic uptake.
INTRODUCTION
Top
Introduction
References
Multiple Vacuolar Functions Are Accompanied by Diverse
Delivery Pathways
The Resident Hydrolase Aminopeptidase I Is Localized to the
Vacuole Independent of the Secretory Pathway
The Aminopeptidase I Propeptide Is Required for Vacuolar
Targeting
-helices (27). The first of
these forms an amphipathic structure; unlike mitochondrial targeting
signals, the charged half is composed of both basic and acidic
residues. Site-specific mutagenesis was used to analyze the location of
targeting information in the API propeptide (28, 29). Small deletions
anywhere within the first helix of the propeptide resulted in a
complete block in targeting. Similar deletions within the second helix
had no affect on targeting; in this case, the altered proteins were
delivered to the vacuole with wild type kinetics. Deletion of the
entire second helix, however, blocks API targeting suggesting some role for this part of the precursor protein in the import process.
Biochemical and Genetic Identification of Targeting
Components
Aminopeptidase I Transits to the Vacuole and Imports as a
Dodecamer
Macroautophagic Protein Import Involves a Double Membrane
Vesicle
Biochemical Studies Support a Macroautophagy-like Mechanism for
API Delivery
API Delivery Morphologically Resembles Macroautophagy
Macroautophagy and the Cvt Pathway Display Different
Kinetics
60p, that is no longer able to enter the secretory
pathway (51). The only way for Pho8
60p to be delivered to the
vacuole is by macroautophagy. Vacuolar delivery can be monitored by
following removal of the C-terminal propeptide.
60p was used as a marker for macroautophagy and API as a marker
for the Cvt pathway. Examining vacuolar uptake of these two proteins
under vegetative and starvation conditions revealed differences between
the two pathways (35). API import is rapid and complete under both
conditions. The kinetics of import are essentially identical in rich or
nitrogen-deficient media. In contrast, uptake of Pho8
60p does not
occur in rich media. Pho8
60p is taken into the vacuole upon
induction of macroautophagy by nitrogen starvation. The kinetics of
uptake, however, are much slower than seen for API, and the yield of
the import process is incomplete; uptake of Pho8
60p plateaus at
approximately 30% import. By three criteria, the conditions of import,
the rate, and the yield, the Cvt pathway and macroautophagy are
distinguishable.
The Machinery Used for API Import Adapts to the Environmental
Conditions
View larger version (30K):
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Fig. 1.
Aminopeptidase I is imported into the
vacuole by a vesicle-mediated process. The precursor protein
oligomerizes in the cytosol and forms complexes that are surrounded by
a sequestering membrane. The environmental conditions that determine
the specific type of vesicle used for import are described in the text.
These double membrane vesicles fuse with the vacuole delivering a
subvacuolar vesicle that is broken down allowing API maturation.
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Conclusions |
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The synthesis and degradation of organelles and macromolecules are controlled in part by the availability of metabolic substrates. If non-fermentable carbon sources, such as glycerol or ethanol, become available, mitochondrial synthesis increases. Similarly, peroxisomes proliferate in the presence of oleic acid or methanol. These organelles are selectively delivered to the vacuole for degradation if glucose or ethanol become available for fermentative or respiratory metabolism, respectively. When yeast cells experience conditions of nitrogen or carbon starvation following growth in rich medium, macroautophagic uptake of bulk cytosol is induced. The turnover of macromolecules and organelles within the vacuole lumen provides the cell with critical building blocks. Tremendous amounts of membrane synthesis, flow, and recycling are involved in these varied biogenesis and degradation events, yet they are not well understood.
There are several possible explanations for the use of so many delivery mechanisms to the yeast vacuole and to the overlap between the Cvt and macroautophagy pathways. First, the vacuole contains numerous hydrolases that appear to have overlapping specificities. Proteolytic capacity is critical for survival under certain environmental conditions. By utilizing multiple targeting pathways for resident hydrolases, the cell ensures at least a partial complement of vacuolar enzymes. Second, the yeast vacuole has functional similarities to the mammalian lysosome, the plant vacuole, and the contractile vacuole of slime mold and other organisms. In higher eukaryotes, the targeting pathways have diverged allowing specific delivery of proteins to each of these compartments. Because yeast maintain these diverse functions within the vacuole, various pathways are needed for transport of the corresponding proteins. Third, the dodecameric nature of API may be critical for its function and/or stability. The oligomeric protein is incapable of entering the vacuole through the secretory pathway and must use an alternate mechanism. The most efficient use of cellular resources is to adapt existing machinery, in this case the macroautophagic pathway, to allow efficient transport of API to the vacuole.
Many questions remain to be answered concerning the various targeting pathways highlighted in this review. The mechanistic functions of the many gene products implicated in the VPS-dependent secretory pathway targeting route have not been established. Relatively few proteins have been identified that are specific for the alternate pathway used by alkaline phosphatase. The degradative pathways for protein and organelle turnover are, in some cases, quite specific. The means by which this specificity is achieved are not known. All of these processes involve membrane flow to the vacuole. The origin of the membranes, the ways in which they form and/or package their cargo, how they are targeted to the vacuole, and whether the phospholipid constituents are recycled are topics that need to be further addressed. Because of these many questions, nonclassical protein targeting to the vacuole remains an exciting field of cell biology research.
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ACKNOWLEDGEMENTS |
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I thank Dr. Sidney Scott and John Kim for critical reading of the manuscript and for many helpful discussions and Dr. Ignacio Sandoval for communicating results prior to publication.
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FOOTNOTES |
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* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This work was supported by the National Institute of General Medical Sciences and the John Simon Guggenheim Memorial Foundation.
This review is dedicated to the memory of Dr. Helmut Holzer.
To whom correspondence should be addressed: Section of
Microbiology, 156 Hutchison Hall, University of California, One Shields Ave., Davis, CA 95616.Tel.: 530-752-0277;Fax: 530-752-9014; E-mail: djklionsky{at}ucdavis.edu.
1 The abbreviations used are: ALP, alkaline phosphatase; API, aminopeptidase I; ER, endoplasmic reticulum; AP, autophagosome; AB, autophagic body.
2 I. Sandoval, personal communication.
3 S. V. Scott and D. J. Klionsky, unpublished data.
4 References 1-8, 13, and 19-21 are reviews.
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REFERENCES4 |
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