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Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Δ cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123–1131, 2001)

Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole1,2. Apg8/Aut7 is an essential factor for autophagy in yeast3,4,5. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus6. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane6. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy6.

Autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. In Saccharomyces cerevisiae, Tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. Here, we show that the protein kinase activity of Apg1 is enhanced by starvation or rapamycin treatment. In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1. This Apg1–Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions. Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

We investigated the enzyme cytochemical localization of sarcosine oxidase (SOX) in the liver and kidney of several mammals using a cerium technique. First we measured the enzyme activities in the liver and kidney of several mammals and in several organs of mice. The highest activity was found in the Chinese hamster, followed by the mouse. Therefore, we used hamster and mouse tissues for enzyme cytochemistry. The liver and kidneys were fixed by perfusion with various concentrations of glutaraldehyde for 10 min. Tissue slices were incubated in reaction medium consisting of 50 mM TRIS-maleate buffer (pH 7.8), 9 mM sodium azide, 9.8 mM sarcosine, 25 microM FAD, 2 mM cerium chloride, 0.002% saponin, and 0.003% Triton X-100 for 0.5-8 h at 37 degrees C. Optimum staining reaction was obtained in tissues fixed with 0.2% glutaraldehyde, followed by incubation for 2-4 h. Electron-dense reaction products were present exclusively in peroxisomes. Within the peroxisomes strong reactions were observed in the matrix subjacent to the limiting membrane decreasing toward the center. The staining reaction was completely inhibited by 2 mM N-bromosuccinimide. These results indicated that SOX is a peroxisomal enzyme and that the enzyme might be associated with the peroxisomal membrane.

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1–localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.

In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507Ashowed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p–Apg5p conjugation.

The vacuole is one of the most prominent compartments in yeast cells. The wild-type yeast cells have a large vacuolar compartment which occupies approximately a quarter of the cell volume, while thevam4 mutant cells exhibit highly fragmented vacuolar morphology. We isolated theVAM4 gene and found that theVAM4 is identical to theYPT7 which encodes a member of small GTP-binding protein superfamily. We introduced mutations to theVAM4/YPT7 which alter nucleotide binding characteristics of the gene product specifically, and their activities for the vacuolar morphogenesis were examined by transforming the mutant genes into yeast cells. The Thr22Asn mutation, which was expected to fix the protein in the GDP-bound state, resulted in loss of function in the vacuolar morphogenesis. Subcellular fractionation analysis indicated that the mutant molecule did not associate with intracellular membranes efficiently. In contrast, Vam4/Ypt7p with the Gln68Leu mutation, which was expected to be the GTP-bound form, complemented the fragmented vacuolar morphology of Δvam4 mutant cells. Vam4/Ypt7p with the Gln68Leu mutation also complemented the defects in the biogenesis of vacuolar alkaline phosphatase whose maturation requires the proper function of Vam4/Ypt7p. Overexpression of the mutant proteins in wild-type cells did not develop dominant-negative effects on the vacuolar assembly. These results indicated that the GTP-bound form of Vam4/Ypt7p promotes the biogenesis and morphogenesis of the yeast vacuolar compartment.

PI-kinase-related protein kinase ATR forms a complex with ATRIP and plays pivotal roles in maintaining genome integrity. When DNA is damaged, the ATR-ATRIP complex is recruited to chromatin and is activated to transduce the checkpoint signal, but the precise kinase activation mechanism remains unknown. Here, we show that ATRIP is phosphorylated in an ATR-dependent manner after genotoxic stimuli. The serine 68 and 72 residues are important for the phosphorylation in vivo and are required exclusively for direct modification by ATR in vitro. Using phospho-specific antibody, we demonstrated that phosphorylated ATRIP accumulates at foci induced by DNA damage. Moreover, the loss of phosphorylation does not lead to detectable changes in the relocalization of ATRIP to nuclear foci nor in the activation of downstream effector proteins. Collectively, our results suggest that the ATR-mediated phosphorylation of ATRIP at Ser-68 and -72 is dispensable for the initial response to DNA damage.

ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR.

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.

Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation1,2,3,4. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae5 and examined the autophagic process at the molecular level6,7,8,9. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.

Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region. We found that overproduction of Apg14p partially reversed the defect in autophagy induced by the apg6-1 mutation. The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole. However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-1 mutant, nor did the apg14 null mutation affect the CPY sorting pathway. Structural analysis of APG6 revealed that APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late-Golgi from the endosome (Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J. Cell Biol. 137, 79-92). Subcellular fractionation indicated that Apg14p and Apg6p peripherally associated with a membrane structure(s). Apg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex. These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway. Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway.

The buoyant densities of the yeast cells of defective vacuolar morphology mutants were examined by equilibrium sedimentation centrifugation in a Percoll density gradient. These vacuoleless mutants also show density fluctuation as wild-type cells during the cell cycle. This suggests that morphological changes of the vacuole are not related to cyclic density fluctuation in Saccharomyces cerevisiae.

Calcium-activated neutral proteases (CANPs) with high sensitivity (microCANP) and low sensitivity (mCANP) to calcium ions were purified individually from rabbit skeletal muscle and rabbit lung and compared as to their electrophoretic properties, calcium requirements and peptide mapping of fragments produced by S. aureus V8 protease digestion of separated subunits. All of the results suggested that there is no difference between the microCANPs as well as between the mCANPs obtained from the two tissues, with respect to the chemical and enzymatic properties. However, the contents of CANPs in these tissues were different.

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the promoter regions of the malB divergent operons was determined. The region of the proximal gene, malE of the malEFG operon, was identified on the basis of the known amino acid sequence of the precursor molecule of maltose-binding protein. The region of malK, the proximal gene of the malKlamB operon, was deduced from the observation that a cloned segment contains an amino-terminal portion of the malK gene. The non-coding region between malE and malK is 299 base pairs long and contains two long GC clusters. Another feature of this region that may be related to the regulation of gene expression is the presence of two palindromic structures between the GC clusters. The DNA regions binding to cyclic AMP binding protein were determined by a method using polyacrylamide gel electrophoresis. The sites are thought to be located close to GC clusters.

1. The low-calcium-requiring form (μCANP) and the high-calcium-requiring form (mCANP) of the calcium-activated neutral proteases were purified to near homogeneity from rabbit cardiac muscle. 2. Each of them was compared with the counterpart of skeletal muscle in respect to subunit composition, calcium sensitivity, pH dependency of the activity and peptide map of the fragments produced byS. aureus V8 protease digestion. 3. All results suggested that mCANP and μCANP from cardiac muscle were almost indistinguishable in various properties with mCANP and μCANP of skeletal muscle, respectively, showing the lack of tissue-specificity of CANPs among these two tissues. However, the total and the relative contents of mCANP and μCANP were different among them.