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Daniel J Klionsky

Publications and source records attributed to Daniel J Klionsky.

At least 19 recordsLinked to original sources
PubMed2007

Methods for monitoring autophagy from yeast to human.

The increasing interest in autophagy in a wide range of organisms, accompanied by an ever-growing influx of researchers into this field, necessitates a good understanding of the methodologies available to monitor this process. In this review we discuss current approaches that can be used to follow the overall process of autophagy, as well as individual steps, from yeast to human. The majority of the review considers methods that apply to macroautophagy; however, we also consider alternative types of degradation including chaperone-mediated autophagy and microautophagy. This information is meant to provide a resource for newcomers as well as a stimulus for experienced researchers who may be prompted to develop additional assays to examine autophagy-related pathways.

Animals↗
PubMed2007

Permeases recycle amino acids resulting from autophagy.

Autophagy is a major survival mechanism for eukaryotes to recycle cellular nutrients during stress conditions (such as nutrient limitation, or the accumulation of damaged organelles). We recently revealed a molecular mechanism by which Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Atg22 is not directly required for autophagic body breakdown, in contrast to previously reported data. Instead, we found that Atg22, Avt3 and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagy.

Amino Acid Transport Systems↗
PubMed2007

Endoplasmic reticulum stress: a new pathway to induce autophagy.

Autophagy is a response to the stress of nutrient limitation in yeast, whereby cytosolic long-lived proteins and organelles are nonselectively degraded, and the resulting macromolecules are recycled to allow new protein synthesis that is essential for survival. We recently revealed that endoplasmic reticulum (ER) stress induces autophagy. When misfolded proteins accumulate in the ER the resulting stress activates the unfolded protein response (UPR) to induce the expression of chaperones and proteins involved in the recovery process. Under conditions of ER stress, the preautophagosomal structure is assembled, and transport of autophagosomes to the vacuole is stimulated in an Atg protein-dependent manner. Interestingly, Atg1 has high kinase activity during ER stress-induced autophagy similar to the situation in starvation-induced autophagy.

Autophagy↗
PubMed2007

Atg26 is not involved in autophagy-related pathways in Saccharomyces cerevisiae.

Autophagy is a degradative pathway conserved among eukaryotes. It is a major route for degradation of long-lived proteins and entire organelles, such as peroxisomes. Atg26, a sterol glucosyltransferase, is specifically required for micro- and macropexophagy, but not for starvation-induced bulk autophagy in Pichia pastoris. Here we study the requirement of Saccharomyces cerevisiae Atg26 in the Cvt pathway, nonspecific autophagy and pexophagy. Our results show that the S. cerevisiae atg26Delta strain is not defective in prApe1 maturation, macroautophagy or peroxisome degradation, in contrast to the situation seen in Pichia pastoris. These studies highlight the importance of examining mutants in multiple organisms.

Autophagy↗
PubMed2006

Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast.

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy. To better understand the mechanisms involved in Atg9 cycling, we performed a yeast two-hybrid-based screen and identified a peripheral membrane protein, Atg11, that interacts with Atg9. We show that Atg11 governs Atg9 cycling through the PAS during specific autophagy. We also demonstrate that the integrity of the actin cytoskeleton is essential for correct targeting of Atg11 to the PAS. We propose that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS that is dependent on the actin cytoskeleton during yeast vegetative growth.

Actins↗
PubMed2006

Aup1p, a yeast mitochondrial protein phosphatase homolog, is required for efficient stationary phase mitophagy and cell survival.

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.

Autophagy↗
PubMed2006

Atg27 is required for autophagy-dependent cycling of Atg9.

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.

Amino Acid Sequence↗
PubMed2006

Atg22 recycles amino acids to link the degradative and recycling functions of autophagy.

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.

Amino Acid Transport Systems↗
PubMed2006

Autophagy in organelle homeostasis: peroxisome turnover.

When cells are confronted with an insufficient supply of nutrients in their extracellular fluid, they may begin to cannibalize some of their internal proteins as well as whole organelles for reuse in the synthesis of new components. This process is termed autophagy and it involves the formation of a double-membrane structure within the cell, which encloses the material to be degraded into a vesicle called an autophagosome. The autophagosome subsequently fuses with a lysosome/vacuole whose hydrolytic enzymes degrade the sequestered organelle. Degradation of peroxisomes is a specific type of autophagy, which occurs in a selective manner and has been mostly studied in yeast. Recently, it was reported that a similar selective process of autophagy occurs in mammalian cells with proliferated peroxisomes. Here we discuss characteristics of the autophagy of peroxisomes in mammalian cells and present a comprehensive model of their likely mechanism of degradation on the basis of known and common elements from other systems.

Animals↗
PubMed2006

Endoplasmic reticulum stress triggers autophagy.

Eukaryotic cells have evolved strategies to respond to stress conditions. For example, autophagy in yeast is primarily a response to the stress of nutrient limitation. Autophagy is a catabolic process for the degradation and recycling of cytosolic, long lived, or aggregated proteins and excess or defective organelles. In this study, we demonstrate a new pathway for the induction of autophagy. In the endoplasmic reticulum (ER), accumulation of misfolded proteins causes stress and activates the unfolded protein response to induce the expression of chaperones and proteins involved in the recovery process. ER stress stimulated the assembly of the pre-autophagosomal structure. In addition, autophagosome formation and transport to the vacuole were stimulated in an Atg protein-dependent manner. Finally, Atg1 kinase activity reflects both the nutritional status and autophagic state of the cell; starvation-induced autophagy results in increased Atg1 kinase activity. We found that Atg1 had high kinase activity during ER stress-induced autophagy. Together, these results indicate that ER stress can induce an autophagic response.

Autophagy↗
PubMed2006

Atg9 sorting from mitochondria is impaired in early secretion and VFT-complex mutants in Saccharomyces cerevisiae.

In eukaryotic cells, the turnover of long-lived proteins and large cytoplasmic structures is mediated by autophagy. Components that have to be eliminated are sequestered into double-membrane vesicles called autophagosomes and delivered into the lysosome or vacuole where they are destroyed by resident hydrolases. The integral membrane protein Atg9 is essential for both autophagy and the cytoplasm-to-vacuole targeting pathway, a selective biosynthetic process in Saccharomyces cerevisiae that is mechanistically and morphologically similar to autophagy. Atg9 cycles between the pre-autophagosomal structure, the putative site of double-membrane vesicle biogenesis and mitochondria. To understand the function of Atg9, and also its trafficking mode between these two locations, we identified mutants that affect specific Atg9 transport steps. We recently reported that five Atg proteins and phosphatidylinositol-3-phosphate regulate Atg9 recycling from the pre-autophagosomal structure. Here, we describe a different category of mutants that blocks Atg9 sorting from mitochondria. All mutants have been previously shown to be required for the normal progression of both the Cvt pathway and autophagy, but their precise role in these transport routes was unknown.

Actins↗
PubMed2006

Autophagy and neurodegeneration.

Autophagy mediates the bulk degradation of cytosolic proteins and organelles. Recent studies using neuron-specific knockout mouse models demonstrate that autophagy deficiency leads to protein aggregation and neurodegeneration, even in the absence of disease-related aggregate-prone proteins.

Animals↗
PubMed2006

Atg11 directs autophagosome cargoes to the PAS along actin cables.

For more than 40 years, autophagy has been almost exclusively studied as a cellular response that allows adaptation to starvation situations. In nutrient-deprived conditions, cytoplasmic components and organelles are randomly sequestered into double-membrane vesicles called autophagosomes, creating the notion that this pathway is a nonselective process (reviewed in Refs 1, 2). Recent results, however, have demonstrated that under certain circumstances, cargoes such as protein complexes, organelles and bacteria can be selectively and exclusively incorporated into double-membrane vesicles.(1) We have recently shown that actin plays an essential role in two selective types of autophagy in the yeast Saccharomyces cerevisiae, the cytoplasm to vacuole targeting (Cvt) pathway and pexophagy, raising the possibility that the structures formed by polymers of this protein helps autophagosomes in recognizing the cargoes that must be delivered to the vacuole.(3) In this addendum, we discuss the possible central role of Atg11 as a molecule connecting cargoes, actin and pre-utophagosomal structure (PAS) elements.

Actins↗
PubMed2006

A systems biology approach to learning autophagy.

With its relevance to our understanding of eukaryotic cell function in the normal and disease state, autophagy is an important topic in modern cell biology; yet, few textbooks discuss autophagy beyond a two- or three-sentence summary. Here, we report an undergraduate/graduate class lesson for the in-depth presentation of autophagy using an active learning approach. By our method, students will work in small groups to solve problems and interpret an actual data set describing genes involved in autophagy. The problem-solving exercises and data set analysis will instill within the students a much greater understanding of the autophagy pathway than can be achieved by simple rote memorization of lecture materials; furthermore, the students will gain a general appreciation of the process by which data are interpreted and eventually formed into an understanding of a given pathway. As the data sets used in these class lessons are largely genomic and complementary in content, students will also understand first-hand the advantage of an integrative or systems biology study: No single data set can be used to define the pathway in full-the information from multiple complementary studies must be integrated in order to recapitulate our present understanding of the pathways mediating autophagy. In total, our teaching methodology offers an effective presentation of autophagy as well as a general template for the discussion of nearly any signaling pathway within the eukaryotic kingdom.

Autophagy↗