Overexpression of Autophagy-Related Genes Inhibits Yeast Filamentous Growth

Under conditions of nitrogen stress, the budding yeast S. cerevisiae initiates a cellular response involving the activation of autophagy, an intracellular catabolic process for the degradation and recycling of proteins and organelles. In certain strains of yeast, nitrogen stress also drives a striking developmental transition to a filamentous form of growth, in which cells remain physically connected after cytokinesis. We recently identified an interrelationship between these processes, with the inhibition of autophagy resulting in exaggerated filamentous growth. Our results suggest a model wherein autophagy mitigates nutrient stress, and filamentous growth is responsive to the degree of this stress. Here, we extended these studies to encompass a phenotypic analysis of filamentous growth upon overexpression of autophagy-related (ATG) genes. Specifically, overexpression of ATG1, ATG3, ATG7, ATG17, ATG19, ATG23, ATG24, and ATG29 inhibited filamentous growth. From our understanding of autophagy in yeast, overexpression of these genes does not markedly affect the activity of the pathway; thus, we do not expect that this filamentous growth phenotype is due strictly to diminished nitrogen stress in ATG overexpression mutants. Rather, these results highlight an additional undefined regulatory mechanism linking autophagy and filamentous growth, possibly independent of the upstream nitrogen-sensing machinery feeding into both processes.

Addendum to:

An Interrelationship Between Autophagy and Filamentous Growth in Budding Yeast

J. Ma, R. Jin, X. Jia, C.J. Dobry, L. Wang, F. Reggiori, J. Zhu and A. Kumar

Genetics 2007; In press     

An interrelationship between autophagy and filamentous growth in budding yeast

Over the last 15 years, yeast pseudohyphal growth (PHG) has been the focus of intense research interest as a model of fungal pathogenicity. Specifically, PHG is a stress response wherein yeast cells deprived of nitrogen form filaments of elongated cells. Nitrogen limitation also induces autophagy, a ubiquitous eukaryotic stress response in which proteins are trafficked to the vacuole/lysosome for degradation and recycling. Although autophagy and filamentous growth are both responsive to nitrogen stress, a link between these processes has not been investigated to date. Here, we present several studies describing an interrelationship between autophagy and filamentous growth. By microarray-based expression profiling, we detect extensive upregulation of the pathway governing autophagy during early PHG and find both processes active under conditions of nitrogen stress in a filamentous strain of budding yeast. Inhibition of autophagy results in increased PHG, and autophagy-deficient yeast induce PHG at higher concentrations of available nitrogen. Our results suggest a model in which autophagy mitigates nutrient stress, delaying the onset of PHG; conversely, inhibition of autophagy exacerbates nitrogen stress, resulting in precocious and overactive PHG. This physiological connection highlights the central role of autophagy in regulating the cell's nutritional state and the responsiveness of PHG to that state.     

Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Σ1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth.     

Multifunction of autophagy-related genes in filamentous fungi

Autophagy (macroautophagy), a highly conserved eukaryotic mechanism, is a non-selective degradation process, helping to maintain a balance between the synthesis, degradation and subsequent recycling of macromolecules to overcome various stress conditions. The term autophagy denotes any cellular process which involves the delivery of cytoplasmic material to the lysosome for degradation. Autophagy, in filamentous fungi plays a critical role during cellular development and pathogenicity. Autophagy, like the mitogen-activated protein (MAP) kinase cascade and nutrient-sensing cyclic AMP (cAMP) pathway, is also an important process for appressorium turgor accumulation in order to penetrate the leaf surface of host plant and destroy the plant defense. Yeast, an autophagy model, has been used to compare the multi-valued functions of ATG (autophagy-related genes) in different filamentous fungi. The autophagy machinery in both yeast and filamentous fungi is controlled by Tor kinase and both contain two distinct phosphatidylinositol 3-kinase complexes. In this review, we focus on the functions of ATG genes during pathogenic development in filamentous fungi.     

Insights into the relationship between the proteasome and autophagy in human and yeast cells

In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions.     

Autophagy during conidiation and conidial germination in filamentous fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)-AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

Atg28, a novel coiled-coil protein involved in autophagic degradation of peroxisomes in the methylotrophic yeast Pichia pastoris

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy. We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.     

Atg28, a Novel Coiled-Coil Protein Involved in Autophagic Degradation of Peroxisomes in the Methylotrophic Yeast Pichia pastoris

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy.

We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.     

Characterization of a novel autophagy-specific gene, ATG29

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. atg29Delta 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.     

Autophagy: a role in metal ion homeostasis?

Nutrient limitation is one of the most common forms of stress encountered by microorganisms in the environment. Surviving this stress depends upon a number of integrated responses, one of the most important of which is autophagy. When the filamentous fungus Aspergillus fumigatus becomes nutrient deprived it undergoes two important processes: the developmental pathway for asexual sporulation (conidiation), and a foraging response that promotes the migration of the hyphal tips into new substrate. To determine the contribution of autophagy to these two functions, we disrupted the A. fumigatus atg1 gene. The data reveal that Atg1 is required for wild-type conidiation of A. fumigatus, but only when nitrogen is limiting. Secondly, we demonstrate that metal ion availability limits the extent to which A. fumigatus can grow without a carbon/nitrogen source and that autophagy is necessary for growth under conditions of metal ion deficiency. These findings indicate that autophagy is responsible for maintaining an adequate supply of nitrogen to support conidiophore development, and provide intriguing new evidence that autophagy is linked to metal ion homeostasis.     

Molecular mechanisms of autophagy in plants: Role of ATG8 proteins in formation and functioning of autophagosomes

Autophagy is an efficient way of degradation and removal of unwanted or damaged intracellular components in plant cells. It plays an important role in recycling of intracellular structures (during starvation, removal of cell components formed during plant development or damaged by various stress factors) and in programmed cell death. Morphologically, autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which are essential for the isolation and degradation of cytoplasmic components. Among autophagic (ATG) proteins, ATG8 from the ubiquitinlike protein family plays a key role in autophagosome formation. ATG8 is also involved in selective autophagy, fusion of autophagosome with the vacuole, and some other intracellular processes not associated with autophagy. In contrast to yeasts that carry a single ATG8 gene, plants have multigene ATG8 families. The reason for such great ATG8 diversity in plants remains unclear. It is also unknown whether all members of the ATG8 family are involved in the formation and functioning of autophagosomes. To answer these questions, the identification of the structure and the possible functions of plant proteins from ATG8 family is required. In this review, we analyze the structures of ATG8 proteins from plants and their homologs from yeast and animal cells, interactions of ATG8 proteins with functional ligands, and involvement of ATG8 proteins in different metabolic processes in eukaryotes.     

Proteins involved in microbody biogenesis and degradation in Aspergillus nidulans

Fungal microbodies (peroxisomes) are inducible organelles that proliferate in response to nutritional cues. Proteins involved in peroxisome biogenesis/proliferation are designated peroxins and are encoded by PEX genes. An autophagy-related process, termed pexophagy, is responsible for the selective removal of peroxisomes from the cell. Several genes involved in pexophagy are also required for autophagy and are collectively known as ATG genes. We have re-analysed the Aspergillus nidulans genome for the presence of PEX and ATG genes and have identified a number of previously missed genes. Also, we manually determined the correct intron positions in each identified gene. The data show that in A. nidulans and related fungi the basic set of genes involved in peroxisome biogenesis or degradation are conserved. However, both processes have features that more closely resemble organelle formation/degradation in mammals rather than yeast. Thus, filamentous fungi like A. nidulans are ideal model systems for peroxisome homeostasis in man.     

线粒体自噬基因对酿酒酵母抗氧化性能的影响

线粒体自噬是指细胞通过自噬的机制选择性地清除线粒体的过程,对维持细胞内稳态具有重要作用。为探究线粒体自噬基因对酿酒酵母(Saccharomyces cerevisiae)细胞抗氧化性能的影响,本研究分别构建了线粒体自噬相关基因ATG8、ATG11、ATG32的缺失和过表达菌株,发现在过氧化氢(H₂O₂)胁迫6 h后,过表达ATG8、ATG11基因显著降低了细胞内活性氧(reactive oxygen species,ROS)含量,分别仅为初始状态的61.23%和46.35%,并显著提高了菌株线粒体膜电位(mitochondrial membrane potential,MMP)和腺嘌呤核苷三磷酸(adenosine-triphosphate,ATP)含量,有助于提高菌株的抗氧化性能。另一方面,基因ATG8、ATG11、ATG32的缺失会导致线粒体损伤及细胞活力显著下降,同时造成胞内ROS失衡,H₂O₂胁迫6 h后,其胞内ROS含量显著升高至初始状态的174.27%、128.68%和200.92%。结果表明,ATG8、ATG11和ATG32可能是调控酵母抗氧化能力的潜在靶点。本研究为进一步研究通过调节线粒体自噬提高酵母抗氧化活性提供了新的线索。     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.