(Received for publication, November 7, 1995; and in revised form, January 10, 1996)
From the
When glucose-starved cells are replenished with glucose, the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the yeast lysosome (vacuole) for degradation. The glucose-induced targeting of FBPase to the vacuole for degradation occurs in cells grown under a variety of metabolic conditions. Immunoelectron microscopic studies demonstrate that the uptake of FBPase by the vacuole is mediated in part by an autophagic process. FBPase can be found on the vacuolar membrane and also at the sites of membrane invaginations. Furthermore, FBPase is associated with different forms of vesicles, which are induced to accumulate inside the vacuole. We have identified peroxisomes as the organelles that are delivered to the vacuole for degradation when cells are replenished with glucose. Ultrastructural studies indicate that peroxisomes are engulfed by the vacuole by an autophagic process, leading to the destruction of whole organelles in the vacuole. Furthermore, the galactose transporter (Gal2p) is also delivered from the plasma membrane to the vacuole for degradation in response to glucose. Gal2p is delivered to the vacuole through the endocytic pathway, as mutants defective in receptor-mediated endocytosis fail to degrade Gal2p in response to glucose.
Many pathways of protein degradation have been identified(1, 2, 3) . Most short-lived normal proteins and abnormal proteins are degraded in the cytosolic, ubiquitin-dependent, or proteasome-dependent pathway(1, 2, 3) , whereas most long-lived proteins are degraded in lysosomes(4, 5) . Lysosomal degradation of cytosolic proteins is regulated by hormones and growth factors and is increased when cells are starved of nutrients(4, 5, 6) . The enhanced lysosomal protein degradation requires the heat shock hsc73 protein and a targeting sequence present in many cytosolic proteins that are targeted to lysosomes for degradation such as RNase A and glyceraldehyde-3-phosphate dehydrogenase(7, 8, 9, 10) . Cytosolic proteins can also be taken up by lysosomes by non-selective autophagy (4, 5, 6) .
The yeast vacuole
plays an important role in the degradation of many cellular
proteins(11, 12, 13) . However, the mechanism
responsible for the uptake of cytosolic proteins into the yeast vacuole
is less understood. It was reported that the key regulatory enzyme in
the gluconeogenesis pathway, fructose-1,6-bisphosphatase (FBPase), ()is inactivated in response to
glucose(14, 15, 16, 17, 18) .
In addition to FBPase, many enzymes involved in the metabolism of
carbohydrates(14, 19, 21) , the tricarboxylic
acid cycle (20) , the control of flux of
sugars(22, 23, 24) , and the oxidation of
fatty acids (25, 26, 27) are also inactivated
by glucose. These enzymes include phosphoenolpyruvate
carboxykinase(19) , C-malate dehydrogenase (20) ,
trehalose-6-phosphate synthase(21) , trehalose-6-phosphate
phosphatase(21) , galactose transporter(22) , maltose
transporter(23) , and alcohol oxidase and
catalase(25, 26, 27) . Some of these enzymes
are cytosolic, while others are localized to the plasma membrane or in
distinct organelles such as peroxisomes. The induction of these enzymes
during glucose starvation serves to maximize the production of energy.
When cells are replenished with fresh glucose, these enzymes are no
longer needed and are inactivated. Given that a large number of enzymes
involved in different metabolic pathways are inactivated by glucose,
catabolite inactivation appears to play a critical role in the
regulation of metabolism.
Studies using vacuolar proteinase-deficient mutants (pep4) have demonstrated that FBPase is targeted from the cytosol to the vacuole for degradation when glucose-starved cells are replenished with glucose(18) . Using indirect immunofluorescence, we have found that FBPase displays an unusual punctate staining in the vacuole, which coincides with numerous particles that are induced to accumulate in the vacuole as seen by Nomarski microscopy. This prompted us to examine the import process at the ultrastructural level using immunoelectron microscopy. We have found that the uptake of FBPase into the vacuole is mediated by an autophagic process. FBPase can be found at the sites where membrane invaginations occur, indicating that FBPase is internalized by an autophagy of membranes structures. Inside the vacuole, FBPase is associated with different types of membranes, suggesting that other vesicles are also delivered to the vacuole for degradation in response to glucose. We have identified peroxisomes and the galactose transporter that are delivered to the vacuole for degradation when cells are replenished with glucose.
Figure 1: Glucose-induced degradation of FBPase is dependent on the PEP4 gene. Untransformed wild type and pep4 cells were grown under various conditions and transferred to glucose for 45 min. Cells were fixed and processed for immunofluorescence microscopic studies with affinity-purified anti-FBPase antibodies as described(18) . a, wild type cells grown in YPD (2% dextrose) for 24 h and adopted to YPO (2% oleic acid) for an additional 24 h. b, the oleate-grown wild type cells were transferred to glucose for 45 min. c, pep4 cells were grown in oleic acid. d, transferred to glucose for 45 min. e and f, pep4 cells were grown in YP containing 2% galactose for 48 h (e) and transferred to glucose for 45 min (f). g and h, pep4 cells were grown in YP containing 2% pyruvate for 48 h (g) and transferred to glucose for 45 min (h). Left panels, Nomarski images of cells; right panels, fluorescence images of FBPase.
Figure 2: Glucose induces autophagy and massive protein degradation. A, electron micrographs of pep4 cells at t = 0 and 45 min following a transfer to glucose. a, cells were grown in acetate at t = 0 min; b, transferred to glucose for 45 min; c, cells were shifted from galactose to glucose for 45 min; d, cells were grown in oleate and transferred to glucose for 45 min. b, total cellular proteins were pulse-labeled for 24 h and chased for 0-5 h in the presence and absence of glucose (a), in wild type and pep4 cells (b), in the presence and absence of 10 µg/ml cycloheximide (c).
To assess the extent of protein degradation that occurred in the vacuole in response to glucose, we metabolically labeled proteins for 24 h in the glucose-starved cells to preferentially label long-lived proteins that were expressed during glucose starvation. We followed the degradation of long-lived proteins for 0-5 h in the presence of glucose and compared the rates of degradation of these proteins in the absence of glucose. Fig. 2B (a) shows that in the absence of glucose, cells degraded long-lived proteins at a slow rate. In a period of 5 h, only 1.5% degradation rate was observed. The addition of glucose accelerated the degradation rate 16-fold. The degradation rate increased linearly and reached 25% in 5 h (Fig. 2B, a). The induced degradation of long-lived proteins occurred primarily in the vacuole, as pep4 mutants blocked the degradation of long-lived proteins to 30% of the wild type level (Fig. 2B, b). In a chase of 5 h in glucose, only 7% of long-lived proteins were degraded in pep4 cells (Fig. 2B, b). Since the vacuole contains a variety of proteinases and that the deletion of the PEP4 gene affects only a subset of proteinases, the contribution of the vacuole could be greater than 70%. Bulk degradation of proteins requires the synthesis of new proteins, as cycloheximide inhibited the degradation of total long-lived proteins in the presence of glucose (Fig. 2B, c).
Figure 3: Uptake of FBPase is mediated by an autophagic process. Immunoelectron microscopic studies with affinity-purified anti-FBPase were performed as described under ``Experimental Procedures.'' a, immunogold labeling of FBPase in the cytosol in pep4 cells at t = 0 min; b, FBPase is in the vacuole in pep4 cells transferred to glucose for 45 min; c and d, higher magnification of the vacuole. Triangles indicate the association of FBPase with the vacuolar membrane. Arrows indicate the staining of FBPase at the sites of membrane invaginations and the association of FBPase with autophagic vesicles.
Figure 4: Peroxisomes are degraded in the vacuole by autophagy. A, pulse-chase experiments were performed in wild type and pep4 cells. Degradation of peroxisomes and mitochondria were followed after a transfer of cells to glucose for 0, 1, 2, 3, and 4 h with anti-peroxisomal thiolase antibodies and anti-mitochondrial hsp70 antibodies, respectively. B, pulse-chase and immunoprecipitation experiments were performed. Proteins were quantified by a PhosphorImager (Molecular Dynamics) using t = 0 as 100%. a, degradation of peroxisomes in wild type and pep4 cells; b, in the presence and absence of 10 µg/ml cycloheximide. C, immunoelectron microscopic localization of peroxisomes in pep4 cells transferred to glucose for 45 min. a, peroxisomes are inside the vacuole in pep4 cells; b and c, peroxisomes are engulfed by the vacuole (arrows).
The kinetics of the degradation of peroxisomes was different from that of FBPase. In wild type cells, FBPase is degraded with a half-life of 30 min in response to glucose. In addition, no FBPase can be detected after 1 h of shift to glucose. Fig. 4B shows that the kinetics of the degradation of peroxisomes was slower than that of FBPase. Peroxisomes were degraded with a half-life of 1.5 h in wild type cells (Fig. 4B, a). Furthermore, 30% of peroxisomal proteins remained after 5 h of chase in glucose (Fig. 4B, a). The significance of these differences is not clear. The remaining peroxisomes may be important for the synthesis of new peroxisomes if cells are regrown in a medium that requires the proliferation of the entire organelles. The degradation of peroxisomes required the PEP4 gene, as pep4 cells blocked the degradation of peroxisomes with a prolonged half-life of more than 10 h (Fig. 4B, a). The addition of cycloheximide in the presence of glucose inhibited the degradation of peroxisomes with a half-life of more than 10 h, indicating that the degradation of peroxisomes required the synthesis of new proteins (Fig. 4B, b).
To study whether peroxisomes were delivered to the vacuole for degradation, we performed immunoelectron microscopic studies on thin sections of pep4 cells that have been transferred to glucose for 45 min. Fig. 4C shows that, despite a long half-life, uptake of peroxisomes by the vacuole could be observed at 45 min of glucose shift (Fig. 4C, a-c). Fig. 4C (a) shows that a total of eight peroxisomes were seen in the whole cell, with four localized inside the vacuole and four in the cytoplasm. Peroxisomes were internalized by an autophagic process, as indicated by the localization of peroxisomal thiolase inside membranous structures indistinguishable from the ones in the cytoplasm. If a direct fusion between peroxisomes and the vacuole had occurred, a mixing of the peroxisomal matrix proteins and a distribution of the peroxisomal thiolase in the matrix of the vacuole would be expected. The engulfment of peroxisomes by the vacuolar membrane could be observed in several thin sections of pep4 cells (arrows in Fig. 4C, b and c).
Figure 5: Gal2p is delivered to the vacuole for degradation through endocytosis. A, wild type and pep4 cells were grown in galactose and transferred to glucose for 0-4 h. Degradation of Gal2p was followed with anti-Gal2p antibodies and the plasma membrane ATPase followed with anti-plasma membrane ATPase antibodies. B, degradation of Gal2p was followed in wild type transferred to glucose in the absence of cycloheximide for 0-4 h (a) or in the presence of 10 µg/ml cycloheximide (b). Wild type cells were shifted to sucrose (c), galactose (d), and acetate (e) for 0-4 h. f, wild type cells replenished with glucose for 0-4 h at 37 °C. Degradation of Gal2p was followed in end1 (g), end2 (h), end3 (i), and end4 (j) mutants for 0-4 h at 37 °C. C, the kinetics of Gal2p degradation in response to glucose in wild type and pep4 cells (a), in the absence and presence of cycloheximide (b), and in end3 and end4 cells (c).
The degradation of Gal2p is dependent on the endocytic
pathway. In Saccharomyces cerevisiae, the pheromone
-factor binds to the
-factor receptor on the surface of a
cells and triggers an internalization of the receptor
complex(32, 33, 34) . Early endosomes
containing the internalized receptor complex mature into late
endosomes, which fuse with post-Golgi vesicles and deliver the receptor
complex to the vacuole for
degradation(32, 33, 34) . Mutants defective
in receptor-mediated endocytosis have been identified. These end mutants show temperature sensitivity for growth. At the
non-permissive temperature, they are defective not only in the
endocytosis of
-factor but also the accumulation of lucifer yellow
in the vacuole(32, 33, 34) . end1 is
identical to vps11 and pep5(33, 35) . They contain no recognizable
vacuole(33) . end3 and end4 affect the
internalization step of the endocytic pathway(34) . To
determine whether Gal2p was degraded in the vacuole through the
endocytic pathway, the degradation of Gal2p was examined in end mutants. Fig. 5B shows that Gal2p was degraded in
response to glucose in wild type cells at 37 °C. The kinetics of
Gal2p degradation was slightly faster at 37 °C than at 30 °C in
wild type cells. At the non-permissive temperature, end1, end2, end3, and end4 all showed defects in
degrading Gal2p (Fig. 5B, g-j).
Representative results of the kinetics of Gal2p degradation are shown
in Fig. 5C. In wild type cells, Gal2p was degraded with
a half-life of 1 h after glucose readdition. The degradation of Gal2p
was blocked in pep4 cells with a prolonged half-life of more
than 10 h (Fig. 5C, a). As was the case for
FBPase, peroxisomes, and long-lived proteins, the addition of
cycloheximide inhibited the degradation of Gal2p with a half-life of
longer than 10 h (Fig. 5C, b). Delayed
degradation of Gal2p with half-lives of longer than 10 h was also
observed in end3 and end4 mutants (Fig. 5C, c), indicating that Gal2p was
degraded in the vacuole through the endocytosis pathway.
When glucose-starved cells are replenished with glucose, massive protein degradation is induced. In a period of 5 h, 25% of total long-lived proteins are degraded. The increased degradation occurs primarily in the vacuole as pep4 cells block the induced degradation in response to glucose. During this time, an accumulation of autophagic vesicles inside the vacuole is also observed. As these vesicles display great heterogeneity, different organelles may be delivered to the vacuole for degradation when glucose is added to the medium. Consistent with that observation, we have identified three pathways of protein targeting into the vacuole for degradation using markers of the cytosol, peroxisomes, and the plasma membrane. Fig. 6illustrates that FBPase, peroxisomes, and Gal2p are all targeted to the vacuole for degradation when cells are transferred to glucose. These three pathways of protein degradation share several common features. (a) The degradation of FBPase, peroxisomes, and Gal2p depend on the vacuolar PEP4 gene. (b) The degradation of FBPase, peroxisomes, and Gal2p all occur in a glucose-regulated manner. (c) The synthesis of new proteins is required for the degradation of FBPase(18) , peroxisomes, and Gal2p (this study). (d) The degradation of FBPase(18) , peroxisomes, and Gal2p are all selective.
Figure 6: Selective uptake of FBPase, peroxisomes, and Gal2p by the vacuole. Glucose induces a targeting of FBPase from the cytosol to the vacuole for degradation. FBPase import into the vacuole occurs regardless of the growth conditions. Glucose also induces a delivery of peroxisomes from the cytoplasm to the vacuole for degradation. In addition, the galactose transporter (Gal2p) is delivered from the plasma membrane to the vacuole for degradation in response to glucose. The degradation of Gal2p is dependent on the endocytic pathway. The synthesis of new proteins is required for the degradation of FBPase, peroxisomes, and Gal2p during regrowth of cells in glucose.
The inhibition of cycloheximide on glucose-induced protein degradation suggested that important genes are expressed to regulate protein degradation during regrowth of cells in glucose. To determine whether cycloheximide affected the uptake of FBPase by the vacuole or proteolysis in the organelle, we performed immunofluorescence experiments and studied the localization of FBPase. Fig. 7shows that, in the absence of cycloheximide, FBPase was degraded in wild type cells transferred to glucose for 45 min (Fig. 7, a and b). By contrast, FBPase remained in the cytosol in the cycloheximide-treated cells (Fig. 7, c and d), indicating that cycloheximide inhibited the degradation of FBPase by blocking the entry of FBPase into the vacuole (Fig. 7, c and d). Since FBPase remained in the cytosol in the cycloheximide-treated cells, the newly synthesized proteins might participate in a process prior to the uptake by the vacuole.
Figure 7: FBPase remains in the cytosol in the cycloheximide-treated cells. Immunofluorescence staining of FBPase in wild type cells transferred to glucose for 45 min in the absence (a and b) or presence of cycloheximide (c and d). Cells were processed for immunofluorescence as described. a, wild type cells at t = 0 min; b, wild type cells transferred to glucose for 45 min in the absence of cycloheximide; c, wild type cells at t = 0 min; d, wild type cells transferred to glucose for 45 min in the presence of cycloheximide.
FBPase can be induced to accumulate in the cytosol by growing cells in a variety of metabolic conditions such as acetate, oleate, galactose, or pyruvate. When cells are transferred from these conditions to glucose, FBPase is targeted to the vacuole for degradation. Therefore, FBPase import into the vacuole takes place regardless of growth conditions. Immunoelectron microscopic studies have revealed that FBPase is taken up at least in part by an autophagic process, as evidenced by an association of FBPase at the sites of membrane invaginations and also with the vesicles inside the vacuole. Two types of autophagic processes have been described in mammalian cells(4, 5, 36, 37, 38, 39, 40, 41) . Microautophagy involves an invagination and a pinch off the lysosomal membranes. Cytosolic proteins can be engulfed and taken up in a non-selective manner during the invagination process(36, 37) . Microautophagy is responsible for the basal rate of protein degradation that occurs in lysosomes in fed animals(36, 37) . Macroautophagy is induced when cells are starved of nutrients or amino acids(4, 5, 38, 39, 40, 41) . Macroautophagy is responsible for the induced protein degradation that also occurs in lysosomes during starvation. Autophagic vacuoles (AV) that contain different types of intracellular organelles surrounded by a membranous structure derived from the endoplasmic reticulum are formed in the cytoplasm(40, 41) . During the formation of AV, cytosolic proteins can be engulfed and enclosed into AV in a non-selective manner. These AV then fuse with lysosomes, resulting in the destruction of AV with enclosed cytosolic proteins in lysosomes (40, 41) . Both microautophagy and macroautophagy have been shown to deliver cytosolic proteins to lysosomes in a non-selective fashion(4, 5, 36, 37, 38, 39) . Based on our immunoelectron microscopy observations that FBPase can be found at the sites where membrane invaginations occur, FBPase appears to be taken up by microautophagy at least during the late stage of the internalization and degradation process. However, a fraction of FBPase is associated with electron-dense materials without distinct membranous structures. We consider three possibilities that may explain this observation. (a) FBPase is taken up by microautophagy by an invagination and engulfment of the vacuolar membrane. The vesicles containing FBPase are pinched off and moved into the matrix. The vesicles are subsequently broken by lipase to release FBPase to the vacuolar matrix for further degradation by proteinases in the vacuolar matrix. The staining of FBPase in the matrix may represent the FBPase that has been released to the matrix, but has not yet been degraded by proteinases. (b) FBPase is transported into the lumen of a different type of vesicles. After a fusion of the vesicles with the vacuole, FBPase can be released directly into the lumen of the vacuole for degradation. This may account for the staining of FBPase in the matrix. This model predicts that an intermediate compartment is used to deliver FBPase to the vacuole for degradation. Since no such vesicles can be observed at the 45-min time point, this process may occur at earlier time points. (c) FBPase is taken up by a direct translocation. FBPase may bind to a receptor protein on the vacuolar membrane and is translocated into the lumen of the vacuole for degradation. Unfolding of FBPase by heat shock proteins may occur to facilitate the transport of FBPase to the vacuole. This model would be similar to the uptake of RNase A by lysosomes in mammalian cells(8, 9, 10) .
Peroxisomes are engulfed by the vacuole also by autophagy, resulting in the degradation of whole organelles in the vacuole. In P. pastoris, both microautophagy and macroautophagy can operate to degrade peroxisomes depending on metabolic conditions(26) . Transferring of methyltrophic P. pastoris to glucose induces the degradation of peroxisomes by microautophagy, whereas transferring of methanol-grown cells to ethanol induces the degradation of peroxisomes by macroautophagy(26) . The protein synthesis inhibitor, cycloheximide, blocks the microautophagy induced by glucose, but not macroautophagy induced by ethanol(26) . In our studies, the degradation of peroxisomes is inhibited by cycloheximide. It is likely that peroxisomes are taken up by microautophagy when S. cerevisiae are transferred to glucose. This is consistent with our immunoelectron microscopy studies that peroxisomes are internalized by an engulfment by the vacuole during regrowth of cells in glucose.
It has been reported that FBPase is stabilized in the proteasome mutants(42) . Recent evidence has indicated that the SSV7 gene required to maintain the integrity of the vacuole is identical to the DOA4 gene involved in the proteasome degradation pathway (43, 44) . ssv7 mutant was originally isolated as mutant defective in vacuolar biogenesis(43) . Vacuolar proteins such as CPY is missorted to the cell surface in ssv7 mutants(43) . The stabilization of FBPase in the proteasome mutants may result from an indirect effect of mutations that impair the biogenesis of the vacuole.
Catabolite inactivation of FBPase was described by Gancedo in 1971 (14) . We show evidence that catabolite inactivation of FBPase is mediated by a selective targeting of FBPase to the vacuole for degradation. Catabolite inactivation of Gal2p was reported by Matern and Holzer in 1977(22) . We show that Gal2p is delivered to the vacuole for degradation. Inactivation of peroxisomes mediated by autophagy was reported in H. polymorpha, P. pastoris, and C. boidinii(25, 26, 27) . Consistent with those findings, we demonstrate that peroxisomes are targeted to the vacuole for degradation by autophagy when S. cerevisiae are transferred to glucose. Therefore, protein targeting and degradation by the vacuole are responsible for the glucose-dependent inactivation of these enzymes, and the vacuole degradation plays an important role in the regulation of the flux of sugars and the metabolism of carbohydrates and fatty acids.