Overexpression of Autophagy-Related Genes Inhibits Yeast Filamentous Growth

Department of Molecular, Cellular and Developmental Biology and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
Autophagy (Impact Factor: 11.75). 11/2007; 3(6):604-9. DOI: 10.4161/auto.4784
Source: PubMed


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.

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Available from: Craig J Dobry, Jan 20, 2014
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    • "It should be noted that the affinity tags may perturb protein folding at the carboxy terminus of some of the gene products, but we expect that the majority of genes in this collection (80–90%) should encode fully functional proteins, extrapolating from large-scale protein localization and affinity purification studies (Gavin et al. 2002; Ho et al. 2002; Kumar et al. 2002a; Ghaemmaghami et al. 2003; Huh et al. 2003; Bharucha et al. 2008). To generate overexpression strains for phenotypic analysis of filamentous growth, we introduced the plasmids individually in 96-well format into a diploid strain of the filamentous S1278b genetic background by a modified form of lithium acetate-mediated transformation as described (Kumar et al. 2000, 2002b; Ma et al. 2007a,b; Bharucha et al. 2008; Jin et al. 2008). All transformants were selected on SC 2Ura, and glycerol stock solutions (15% glycerol) were prepared. "
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    ABSTRACT: The budding yeast Saccharomyces cerevisiae can respond to nutritional and environmental stress by implementing a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This growth transition has been studied extensively as a model signaling system with similarity to processes of hyphal development that are linked with virulence in related fungal pathogens. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined. Here, we address the genetic basis of yeast pseudohyphal growth by implementing a systematic analysis of 4,909 genes for overexpression phenotypes in a filamentous strain of S. cerevisiae. Our results identify 551 genes conferring exaggerated invasive growth upon overexpression under normal vegetative growth conditions. This cohort includes 79 genes lacking previous phenotypic characterization. Pathway enrichment analysis of the gene set identifies networks mediating MAPK signaling and cell cycle progression. In particular, overexpression screening suggests that nuclear export of the osmoresponsive mitogen-activated protein kinase (MAPK) Hog1p may enhance pseudohyphal growth. The function of nuclear Hog1p is unclear from previous studies, but our analysis using a nuclear-depleted form of Hog1p is consistent with a role for nuclear Hog1p in repressing pseudohyphal growth. Through epistasis and deletion studies, we also identified genetic relationships with the G2 cyclin Clb2p and phenotypes in filamentation induced by S-phase arrest. In sum, this work presents a unique and informative resource towards understanding the breadth of genes and pathways that collectively constitute the molecular basis of filamentation.
    Genetics 02/2013; 193(4). DOI:10.1534/genetics.112.147876 · 5.96 Impact Factor
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    • "In the fungus Podospora anserina, disruption of genes encoding Atg1 and Atg8 homologs resulted in similar phenotypic changes in growth and differentiation (Pinan-Lucarré et al., 2003). This is consistent with findings from Saccharomyces cerevisiae where disruption of different autophagy genes resulted in similar changes in phenotypic traits, e.g., inhibition of filamentous growth (Ma et al., 2007). "
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    ABSTRACT: 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.
    Fungal Genetics and Biology 01/2009; 46(1-46):1-8. DOI:10.1016/j.fgb.2008.10.010 · 2.59 Impact Factor
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    ABSTRACT: Genomic and proteomic approaches combined with traditional methods in molecular and cell biology have been applied to the model organism Saccharomyces cerevisiae, resulting in the identification of links between nitrogen-response pathways and the discovery of previously overlooked genes in the yeast genome. Pseudohyphal growth and autophagy have been studied separately as nitrogen stress response pathways. Pseudohyphal growth refers to a developmental transition in yeast, wherein nitrogen stress results in the formation of branched and elongated filaments, called pseudohyphae. Autophagy is a stress response induced by conditions of nitrogen deprivation in which proteins are trafficked to the vacuole for degradation and recycling. Our studies using microarray-based expression profiling revealed extensive upregulation of the components within the autophagy pathway during early pseudohyphal growth. While both pathways are activated upon nitrogen stress, the inhibition of autophagy results in increased pseudohyphal growth. This result suggests a model in which autophagy mitigates nutrient stress, delaying the onset of pseudohyphal growth; conversely, inhibition of autophagy exacerbates nitrogen stress, resulting in precocious and overactive pseudohyphal growth. Further phenotypic analysis of pseudohyphal growth upon overexpression of autophagy-related (ATG) genes shows that overexpression of several ATG genes inhibits pseudohyphal growth. Since overexpression of ATG genes does not significantly affect autophagic activity or cellular nitrogen stress, this result suggests that additional undefined regulatory mechanism regulate the interrelationship between these processes. In a separate study, a collection of 276 genes encoding caboxy-terminal fusions to yellow fluorescent protein has been constructed. This plasmid-based collection consisting of genes functioning as kinases, transcription factors and signaling proteins serves as a toolkit for future large-scale localization studies. In the last part of my thesis, the identification and characterization of a novel nested anstisense gene, NAG1, is described. NAG1 is entirely within the coding sequence of YGR031W in an antisense orientation on the opposite strand. Further analysis shows that NAG1 plays a role in cell wall biogenesis and is under control of the cell wall integrity pathway. Beyond its function in cell wall biogenesis, NAG1 is noteworthy in that it represents the first example of a nested protein-coding gene in the yeast genome. Ph.D. Molecular, Cellular, and Developmental Biology University of Michigan, Horace H. Rackham School of Graduate Studies
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