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1.  Molecular mechanism of autophagy in yeast, Saccharomyces cerevisiae. 
Bulk degradation of cytosol and organelles is important for cellular homeostasis under nutrient limitation, cell differentiation and development. This process occurs in a lytic compartment, and autophagy is the major route to the lysosome and/or vacuole. We found that yeast, Saccharomyces cerevisiae, induces autophagy under various starvation conditions. The whole process is essentially the same as macroautophagy in higher eukaryotic cells. However, little is known about the mechanism of autophagy at a molecular level. To elucidate the molecules involved, a genetic approach was carried out and a total of 16 autophagy-defective mutants (apg) were isolated. So far, 14 APG genes have been cloned. Among them we recently found a unique protein conjugation system essential for autophagy. The C-terminal glycine residue of a novel modifier protein Apg12p, a 186-amino-acid protein, is conjugated to a lysine residue of Apg5p, a 294-amino-acid protein, via an isopeptide bond. We also found that apg7 and apg10 mutants were unable to form an Apg12p-Apg5p conjugate. The conjugation reaction is mediated via Apg7p, E1-like activating enzyme and Apg10p, indicating that it is a ubiquitination-like system. These APG genes have mammalian homologues, suggesting that the Apg12 system is conserved from yeast to human. Further molecular and cell biological analyses of APG gene products will give us crucial clues to uncover the mechanism and regulation of autophagy.
PMCID: PMC1692668  PMID: 10582243
2.  The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway 
The Journal of Cell Biology  2000;151(2):263-276.
Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule “X” and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.
PMCID: PMC2192639  PMID: 11038174
autophagy/Cvt pathway; Apg4/Aut2; cysteine protease; ubiquitination; deubiquitination
3.  Apg5p Functions in the Sequestration Step in the Cytoplasm-to-Vacuole Targeting and Macroautophagy Pathways 
Molecular Biology of the Cell  2000;11(3):969-982.
The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5ts. Biochemical analysis of API import in the apg5ts strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in the apg5ts mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.
PMCID: PMC14824  PMID: 10712513
4.  Apg7p/Cvt2p Is Required for the Cytoplasm-to-Vacuole Targeting, Macroautophagy, and Peroxisome Degradation Pathways 
Molecular Biology of the Cell  1999;10(5):1337-1351.
Proper functioning of organelles necessitates efficient protein targeting to the appropriate subcellular locations. For example, degradation in the fungal vacuole relies on an array of targeting mechanisms for both resident hydrolases and their substrates. The particular processes that are used vary depending on the available nutrients. Under starvation conditions, macroautophagy is the primary method by which bulk cytosol is sequestered into autophagic vesicles (autophagosomes) destined for this organelle. Molecular genetic, morphological, and biochemical evidence indicates that macroautophagy shares much of the same cellular machinery as a biosynthetic pathway for the delivery of the vacuolar hydrolase, aminopeptidase I, via the cytoplasm-to-vacuole targeting (Cvt) pathway. The machinery required in both pathways includes a novel protein modification system involving the conjugation of two autophagy proteins, Apg12p and Apg5p. The conjugation reaction was demonstrated to be dependent on Apg7p, which shares homology with the E1 family of ubiquitin-activating enzymes. In this study, we demonstrate that Apg7p functions at the sequestration step in the formation of Cvt vesicles and autophagosomes. The subcellular localization of Apg7p fused to green fluorescent protein (GFP) indicates that a subpopulation of Apg7pGFP becomes membrane associated in an Apg12p-dependent manner. Subcellular fractionation experiments also indicate that a portion of the Apg7p pool is pelletable under starvation conditions. Finally, we demonstrate that the Pichia pastoris homologue Gsa7p that is required for peroxisome degradation is functionally similar to Apg7p, indicating that this novel conjugation system may represent a general nonclassical targeting mechanism that is conserved across species.
PMCID: PMC25275  PMID: 10233148
5.  Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells 
The Journal of Cell Biology  2001;152(4):657-668.
In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein–tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.
PMCID: PMC2195787  PMID: 11266458
autophagy; ubiquitin-like protein; autophagosome; isolation membrane; gene targeting
6.  Membrane Recruitment of Aut7p in the Autophagy and Cytoplasm to Vacuole Targeting Pathways Requires Aut1p, Aut2p, and the Autophagy Conjugation Complex 
The Journal of Cell Biology  2001;152(1):51-64.
Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells survive periods of nutritional starvation. Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p represents an early step in vesicle formation. In this study, we identify several requirements for Aut7p membrane association. After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes. This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.
PMCID: PMC2193654  PMID: 11149920
autophagy; lysosome; protein targeting; vacuole; yeast
7.  Tor-Mediated Induction of Autophagy via an Apg1 Protein Kinase Complex 
The Journal of Cell Biology  2000;150(6):1507-1513.
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.
PMCID: PMC2150712  PMID: 10995454
starvation; Cvt; yeast; rapamycin; phosphorylation
8.  Apg2 Is a Novel Protein Required for the Cytoplasm to Vacuole Targeting, Autophagy, and Pexophagy Pathways* 
The Journal of biological chemistry  2001;276(32):30442-30451.
To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.
PMCID: PMC2737745  PMID: 11382760
9.  Apg9p/Cvt7p Is an Integral Membrane Protein Required for Transport Vesicle Formation in the Cvt and Autophagy Pathways 
The Journal of Cell Biology  2000;148(3):465-480.
In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents.
Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.
PMCID: PMC2174799  PMID: 10662773
aminopeptidase I; lysosome; protein targeting; vacuole; yeast
10.  Cooperative Binding of the Cytoplasm to Vacuole Targeting Pathway Proteins, Cvt13 and Cvt20, to Phosphatidylinositol 3-Phosphate at the Pre-autophagosomal Structure Is Required for Selective Autophagy* 
The Journal of biological chemistry  2002;277(33):30198-30207.
Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative complex composed of Apg1 kinase and several interacting proteins that are specific for either the Cvt pathway or autophagy. A second required complex includes a phosphatidylinositol (PtdIns) 3-kinase and associated proteins that are involved in its activation and localization. The majority of proteins required for the Cvt and autophagy pathways localize to a perivacuolar pre-autophagosomal structure. We show that the Cvt13 and Cvt20 proteins are required for transport of precursor Ape1 through the Cvt pathway. Both proteins contain phox homology domains that bind PtdIns(3)P and are necessary for membrane localization to the pre-autophagosomal structure. Functional phox homology domains are required for Cvt pathway function. Cvt13 and Cvt20 interact with each other and with an autophagy-specific protein, Apg17, that interacts with Apg1 kinase. These results provide the first functional connection between the Apg1 and PtdIns 3-kinase complexes. The data suggest a role for PtdIns(3)P in the Cvt pathway and demonstrate that this lipid is required at the pre-autophagosomal structure.
PMCID: PMC2754692  PMID: 12048214
11.  Chemical Genetic Analysis of Apg1 Reveals A Non-kinase Role in the Induction of Autophagy 
Molecular Biology of the Cell  2003;14(2):477-490.
Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones. In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics. In this system, the induction process modifies a constitutive trafficking pathway called the cytoplasm-to-vacuole targeting (Cvt) pathway, which transports the vacuolar hydrolase aminopeptidase I, from the formation of small Cvt vesicles to the formation of autophagosomes. Apg1 is one of the proteins required for the direct signal transduction cascade that modifies membrane dynamics. Although Apg1 is required for both the Cvt pathway and autophagy, we find that Apg1 kinase activity is required only for Cvt trafficking of aminopeptidase I but not for import via autophagy. In addition, the data support a novel role for Apg1 in nucleation of autophagosomes that is distinct from its catalytic kinase activity and imply a qualitative difference in the mechanism of autophagosome and Cvt vesicle formation.
PMCID: PMC149986  PMID: 12589048
12.  Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast 
The Journal of Cell Biology  1999;147(2):435-446.
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.
PMCID: PMC2174223  PMID: 10525546
autophagy; autophagosome; Apg8/Aut7p; Saccharomyces cerevisiae; vacuole
13.  Two Distinct Vps34 Phosphatidylinositol 3–Kinase Complexes Function in Autophagy and Carboxypeptidase Y Sorting inSaccharomyces cerevisiae 
The Journal of Cell Biology  2001;152(3):519-530.
Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3–kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3–kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3–kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3–kinase complex(es). We propose that multiple Vps34p–Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.
PMCID: PMC2196002  PMID: 11157979
Phosphatidylinositol 3–kinase; autophagy; Vps34p; Vps30p/Apg6p; CPY sorting
14.  Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation* 
The Journal of biological chemistry  2001;277(1):763-773.
Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.
PMCID: PMC2754695  PMID: 11675395
15.  Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein 
Molecular Biology of the Cell  1999;10(5):1353-1366.
Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25–30 kDa protein. This conjugate was not observed in cells expressing Gsa7(ΔATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.
PMCID: PMC25277  PMID: 10233149
16.  Cvt9/Gsa9 Functions in Sequestering Selective Cytosolic Cargo Destined for the Vacuole 
The Journal of Cell Biology  2001;153(2):381-396.
Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.
We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.
PMCID: PMC2169458  PMID: 11309418
autophagy; degradation; lysosome; peroxisome; vacuole
17.  Nogo-A couples with Apg-1 through interaction and co-ordinate expression under hypoxic and oxidative stress 
Biochemical Journal  2013;455(Pt 2):217-227.
Nogo-A is the largest isoform of the Nogo/RTN4 (reticulon 4) proteins and has been characterized as a major myelin-associated inhibitor of regenerative nerve growth in the adult CNS (central nervous system). Apart from the myelin sheath, Nogo-A is expressed at high levels in principal neurons of the CNS. The specificity of Nogo-A resides in its central domain, NiG. We identified Apg-1, a member of the stress-induced Hsp110 (heat-shock protein of 110 kDa) family, as a novel interactor of NiG/Nogo-A. The interaction is selective because Apg-1 interacts with Nogo-A/RTN4-A, but not with RTN1-A, the closest paralogue of Nogo-A. Conversely, Nogo-A binds to Apg-1, but not to Apg-2 or Hsp105, two other members of the Hsp110 family. We characterized the Nogo-A–Apg-1 interaction by affinity precipitation, co-immunoprecipitation and proximity ligation assay, using primary hippocampal neurons derived from Nogo-deficient mice. Under conditions of hypoxic and oxidative stress we found that Nogo-A and Apg-1 were tightly co-regulated in hippocampal neurons. Although both proteins were up-regulated under hypoxic conditions, their expression levels were reduced upon the addition of hydrogen peroxide. Taken together, we suggest that Nogo-A is closely involved in the neuronal response to hypoxic and oxidative stress, an observation that may be of relevance not only in stroke-induced ischaemia, but also in neuroblastoma formation.
The nerve growth inhibitor Nogo-A selectively binds to the heat-shock protein Apg-1 and the expression levels of these two interactors are co-regulated under different forms of stress in neurons.
PMCID: PMC3806365  PMID: 23909438
heat-shock protein; hypoxia; interaction; neuron; Nogo; oxidative stress; BiP, immunoglobulin heavy-chain-binding protein; CHO, Chinese-hamster ovary; CNS, central nervous system; ER, endoplasmic reticulum; EBFP2, enhanced blue fluorescent protein 2; Hsp, heat-shock protein; PFA, paraformaldehyde; PLA, proximity ligation assay; Prdx2, peroxiredoxin 2; RHD, reticulon homology domain; ROS, reactive oxygen species; RTN, reticulon; TCA, trichloroacetic acid
18.  Cvt19 Is a Receptor for the Cytoplasm-to-Vacuole Targeting Pathway 
Molecular cell  2001;7(6):1131-1141.
Cvt19 is specifically required for the transport of resident vacuolar hydrolases that utilize the cytoplasm-to-vacuole targeting (Cvt) pathway. Autophagy (Apg) and pexophagy, processes that use the majority of the same protein components as the Cvt pathway, do not require Cvt19. Cvt19GFP is localized to punctate structures on or near the vacuole surface. Cvt19 is a peripheral membrane protein that binds to the precursor form of the Cvt cargo protein aminopeptidase I (prAPI) and travels to the vacuole with prAPI. These results suggest that Cvt19 is a receptor protein for prAPI that allows for the selective transport of this protein by both the Cvt and Apg pathways.
PMCID: PMC2767243  PMID: 11430817
19.  Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase 
Journal of molecular biology  2013;425(22):4167-4176.
Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.
PMCID: PMC3894629  PMID: 23876706
DNA damage; translesion DNA replication; polycyclic aromatic hydrocarbons; Y family polymerase; environmental pollution
20.  The Ccz1-Mon1 Protein Complex Is Required for the Late Step of Multiple Vacuole Delivery Pathways* 
The Journal of biological chemistry  2002;277(49):47917-47927.
Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δ and ccz1Δ strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δ and mon1Δ are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion.
PMCID: PMC2754690  PMID: 12364329
21.  Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p 
Molecular and Cellular Biology  2001;21(17):5742-5752.
In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.
PMCID: PMC87294  PMID: 11486014
22.  Processing of a Pestivirus Protein by a Cellular Protease Specific for Light Chain 3 of Microtubule-Associated Proteins 
Journal of Virology  2004;78(11):5900-5912.
The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme. The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.
PMCID: PMC415803  PMID: 15140988
23.  Autophagosome Requires Specific Early Sec Proteins for Its Formation and NSF/SNARE for Vacuolar Fusion 
Molecular Biology of the Cell  2001;12(11):3690-3702.
Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.
PMCID: PMC60286  PMID: 11694599
24.  Anticardiolipin in Porphyromonas gingivalis Antisera Causes Fetal Loss in Mice 
Journal of Dental Research  2013;92(9):814-818.
β2-glycoprotein I (β2GPI)-dependent anticardiolipin autoantibodies (aCl) are associated with thrombosis and fetal loss. Some microbial pathogens can induce pathogenic antibodies cross-reactive with β2GPI. Sera from a significant percentage of periodontitis patients contain aCl, and some periodontal pathogens contain antigens with peptide sequences having homology to β2GPI. We hypothesized that antibodies raised against P. gingivalis (aPg) contain pathogenic aCl that induce fetal resorption. We immunized mice with β2GPI, P. gingivalis W83, or an arg-gingipain-defective mutant of P. gingivalis (HF18). IgG fractions of aPg were immunoabsorbed to remove aCl-like antibodies (abs-aPg). IgG fractions were administered intravenously into tail veins of mated BALB/c females at day 0 of pregnancy. At day 15, the proportions of fetal resorptions were evaluated. The prevalence of fetal loss was significantly greater in the aPg group than in the control IgG group (21.2% vs. 5.3%, p = .001), and greater in the aPg group than in the abs-aPg group (21.2% vs. 12%, p < .05). There were no fetal resorptions observed in the aPgHF18 group (p = .0005 compared with aPg, p = .17 compared with control). aPg antibody contains activity consistent with pathogenic aCl, and the antigen inducing the antibodies that cause increased fetal loss may be on the arg-gingipain protease of P. gingivalis.
PMCID: PMC3744271  PMID: 23857644
periodontal diseases; antibodies; pregnancy; fetus; molecular mimicry; mice
25.  Antibacterial Effect of Autologous Platelet-Rich Gel Derived from Subjects with Diabetic Dermal Ulcers In Vitro 
Journal of Diabetes Research  2013;2013:269527.
Background. Autologous platelet-rich gel (APG) is an effective method to improve ulcer healing. However, the mechanisms are not clear. This study aimed to investigate the antibacterial effect of APG in vitro. Methods. Platelet-rich plasma (PRP), platelet-poor plasma (PPP) and APG were prepared from whole blood of sixteen diabetic patients with dermal ulcers. Antibacterial effects against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were evaluated by bacteriostasis assay of APG, PRP, and APG-APO (APG combined with apocynin), with phosphate-buffered saline (PBS) and PPP as the control group. Results. (1) Compared to the PBS and PPP, the APG and APG-APO groups showed strong antibacterial activity against Staphylococcus aureus. There was no significant difference (P > 0.05) between APG and APG-APO. (2) Compared to PBS, APG, APG-APO, and PRP showed obvious antibacterial effects against Escherichia coli and Pseudomonas aeruginosa. No significant difference (P > 0.05) was revealed among the three groups. Compared to the PPP group, they did not show antibacterial effect against Escherichia coli and Pseudomonas aeruginosa (P > 0.05). Conclusions. APG has antibacterial effect against Staphylococcus aureus mediated by platelet activation in the diabetic patients with dermal ulcer, and does not present obvious antibacterial effect against Escherichia coli or Pseudomonas aeruginosa. Combination of APG and antibiotics may have synergistic antibacterial effect.
PMCID: PMC3647554  PMID: 23671863

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