Mitophagy in yeast: known unknowns and unknown unknowns.

Mitophagy, the autophagic breakdown of mitochondria, is observed in eukaryotic cells under various different physiological circumstances. These can be broadly categorized into two types: mitophagy related to quality control events and mitophagy induced during developmental transitions. Quality control mitophagy involves the lysosomal or vacuolar degradation of malfunctioning or superfluous mitochondria within lysosomes or vacuoles, and this is thought to serve as a vital maintenance function in respiring eukaryotic cells. It plays a crucial role in maintaining physiological balance, and its disruption has been associated with the progression of late-onset diseases. Developmentally induced mitophagy has been reported in the differentiation of metazoan tissues which undergo metabolic shifts upon developmental transitions, such as in the differentiation of red blood cells and muscle cells. Although the mechanistic studies of mitophagy in mammalian cells were initiated after the initial mechanistic findings in Saccharomyces cerevisiae, our current understanding of the physiological role of mitophagy in yeast remains more limited, despite the presence of better-defined assays and tools. In this review, I present my perspective on our present knowledge of mitophagy in yeast, focusing on physiological and mechanistic aspects. I aim to focus on areas where our understanding is still incomplete, such as the role of mitochondrial dynamics and the phenomenon of protein-level selectivity.

Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation.

Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.

New gadget in the membrane trafficking toolbox: A novel inhibitor of SNARE priming.

NSF (N-ethylmaleimide sensitive factor) and its yeast counterpart Sec18 are highly conserved homohexameric proteins that play vital roles in eukaryotic membrane trafficking. Sec18 functions by disrupting SNARE complexes formed in cis, on the same membrane. However, the molecular mechanisms of this process are poorly understood, in large part due to the lack of selective, reversible inhibitors. A new study by Sparks et al. now reports a small molecule that appears to selectively inhibit Sec18 action in an in vitro assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming.

Cardiolipin Regulates Mitophagy through the Protein Kinase C Pathway.

Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, is important for cardiovascular health, and perturbation of CL metabolism is implicated in cardiovascular disease. Although the role of CL in mitochondrial function, biogenesis, and genome stability has been studied, recent findings indicate that it is essential for functions apart from mitochondrial bioenergetics. In this study, we report that mitophagy is perturbed in CL-deficient yeast cells. Mutants of autophagy/mitophagy genes ATG8, ATG18, and ATG32 synthetically interact with CL synthase mutant crd1Δ. CL-deficient cells exhibited decreased GFP-tagged mitochondrial proteins inside the vacuole and decreased free GFP, consistent with decreased mitophagy. Both PKC and high osmolarity glycerol (HOG) MAPK pathways were shown previously to be required for mitophagy. Activation of both MAPKs was defective in CL-deficient cells. Deletion of HOG pathway genes SHO1, SSK1, STE50, and HOG1 exacerbated crd1Δ growth. 1 m sorbitol and 0.2 m NaCl, which induce the HOG pathway, rescued growth of the mutant. Activation of the MAPK Slt2p was defective in crd1Δ cells, and up-regulation of the PKC pathway by expression of the PKC1R398P gene, which encodes constitutively activated Pkc1p, rescued crd1Δ growth and mitophagy defects. These findings indicate that loss of CL impairs MAPK pathway activation, and decreased activation of the PKC pathway leads to defective mitophagy.

Roles of mitophagy in cellular physiology and development.

The autophagic degradation of mitochondria, or mitophagy, has been shown to occur in eukaryotic cells under various physiological conditions. Broadly, these fall into two categories: quality-control related mitophagy and developmentally induced mitophagy. Quality-control related mitophagy, which is the lysosomal/vacuolar degradation of malfunctioning or superfluous mitochondria, is an important housekeeping function in respiring eukaryotic cells. It plays an essential role in physiological homeostasis and its deregulation has been linked to the progression of late-onset diseases. On the other hand, developmental processes such as reticulocyte maturation have also been shown to involve mitophagy. Importantly, there are clear differences between these processes. Unlike our knowledge of the more general degradation of soluble cytosolic content during starvation-induced macroautophagy, the mechanisms involved in the selective autophagic degradation of mitochondria have only recently begun to receive significant attention. Here, we review the current literature on these topics and proceed to provide specific examples from yeast and mammalian systems. Finally, we cover experimental approaches, with a focus on proteomic methods dedicated to the study of mitophagy in different systems.

Mitophagy as a stress response in mammalian cells and in respiring S. cerevisiae.

The degradation of malfunctioning or superfluous mitochondria in the lysosome/vacuole is an important housekeeping function in respiring eukaryotic cells. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy, and plays a role in physiological homoeostasis as well as in the progression of late-onset diseases. Although the mechanism of bulk degradation by macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. In this mini-review, we introduce mitophagy as a form of mitochondrial quality control and proceed to provide specific examples from yeast and mammalian systems. We then discuss the relationship of mitophagy to mitochondrial stress, and provide a broad mechanistic overview of the process with an emphasis on evolutionarily conserved pathways.

On Hill coefficients and subunit interaction energies.

The study of cooperative ligand binding to multimeric proteins aims to explain complex cooperative binding phenomena using concepts derived from ideal binding isotherms. The purpose of such efforts is the dissection of the cooperative binding isotherm into its interacting components, a result with a clear mechanistic value. Historically, cooperative binding is usually quantified using the Hill coefficient, [Formula: see text], defined as the slope of the Hill plot at 50 % saturation. It was previously shown that the slope of the Hill plot throughout the titration is equal to the ratio of the binding variance in the system under study, to the binding variance of a reference non-interacting system. In the present contribution, this leads to a broader approach towards quantifying cooperativity, which empirically links cooperativity to the ensemble average of the subunit interaction energy. The resulting equations can be used to derive average differential subunit interaction energies directly from experimental binding isotherms. Combined with recent experimental advances in assessing binding distributions in multimeric proteins, these equations can also be used to calculate individual subunit interaction energies for specific n-ligated protein species.

Regulation of autophagy by amino acid availability in S. cerevisiae and mammalian cells.

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic organisms analyzed to date. The study of autophagy has exploded over the last decade or so, branching into numerous aspects of cellular and organismal physiology. From basic functions in starvation and quality control, autophagy has expanded into innate immunity, aging, neurological diseases, redox regulation, and ciliogenesis, to name a few roles. In the present review, I would like to narrow the discussion to the more classical roles of autophagy in supporting viability under nutrient limitation. My aim is to provide a semblance of a historical overview, together with a concise, and perhaps subjective, mechanistic and functional analysis of the central questions in the autophagy field.

PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress.

In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity.

Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution.

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.

Identification of gold nanoparticle-resistant mutants of Saccharomyces cerevisiae suggests a role for respiratory metabolism in mediating toxicity.

Positively charged gold nanoparticles (0.8-nm core diameter) reduced yeast survival, but not growth, at a concentration of 10 to 100 µg/ml. Among 17 resistant deletion mutants isolated in a genome-wide screen, highly significant enrichment was observed for respiration-deficient mutants lacking genes encoding proteins associated with the mitochondrion.

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation.

The mitophagic degradation of mitochondrial matrix proteins in Saccharomyces cerevisiae was previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates.

Where is the field of autophagy research heading?

In this editors' corner, the section editors were asked to indicate where they see the autophagy field heading and to suggest what they consider to be key unanswered questions in their specialty area.

Harnessing yeast subcellular compartments for the production of plant terpenoids.

The biologically and commercially important terpenoids are a large and diverse class of natural products that are targets of metabolic engineering. However, in the context of metabolic engineering, the otherwise well-documented spatial subcellular arrangement of metabolic enzyme complexes has been largely overlooked. To boost production of plant sesquiterpenes in yeast, we enhanced flux in the mevalonic acid pathway toward farnesyl diphosphate (FDP) accumulation, and evaluated the possibility of harnessing the mitochondria as an alternative to the cytosol for metabolic engineering. Overall, we achieved 8- and 20-fold improvement in the production of valencene and amorphadiene, respectively, in yeast co-engineered with a truncated and deregulated HMG1, mitochondrion-targeted heterologous FDP synthase and a mitochondrion-targeted sesquiterpene synthase, i.e. valencene or amorphadiene synthase. The prospect of harnessing different subcellular compartments opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Aup1-mediated regulation of Rtg3 during mitophagy.

Mitophagy is an autophagic process that degrades mitochondria by an intracellular engulfment that leads to their delivery into the lumen of the cell's hydrolytic compartment, such as the lysosome in animal cells or the vacuole in yeast. It is hypothesized that such processes serve a quality control function to prevent or slow the accumulation of malfunctioning mitochondria, which are thought in turn to underlie central aspects of the aging process in eukaryotic organisms. We recently identified a conserved mitochondrial protein phosphatase homolog, Aup1, which is required for efficient stationary phase mitophagy in yeast. In the present report, we demonstrate that the retrograde signaling pathway (RTG) is defective in aup1Delta mutants. In agreement with a role for Aup1 in the regulation of the RTG pathway, we find that deletion of RTG3, a transcription factor that mediates the RTG response, causes a defect in stationary phase mitophagy and that deletion of AUP1 leads to changes in Rtg3 phosphorylation patterns under these conditions. In addition, we find that mitophagic conditions lead to induction of RTG pathway target genes in an Aup1-dependent fashion. Thus, our results suggest that the function of Aup1 in mitophagy could be explained through its regulation of Rtg3-dependent transcription.

Identification of rose phenylacetaldehyde synthase by functional complementation in yeast.

Rose flowers, like flowers and fruits of many other plants, produce and emit the aromatic volatiles 2-phenylacetaldehyde (PAA) and 2-phenylethylalchohol (PEA) which have a distinctive flowery/rose-like scent. Previous studies in rose have shown that, similar to petunia flowers, PAA is formed from L: -phenylalanine via pyridoxal-5'-phosphate-dependent L: -aromatic amino acid decarboxylase. Here we demonstrate the use of a Saccharomyces cerevisiae aro10 mutant to functionally characterize a Rosa hybrida cv. Fragrance Cloud sequence (RhPAAS) homologous to petunia phenylacetaldehyde synthase (PhPAAS). Volatile headspace analysis of the aro10 knockout strain showed that it produces up to eight times less PAA and PEA than the WT. Expression of RhPAAS in aro10 complemented the yeast's mutant phenotype and elevated PAA levels, similar to petunia PhPAAS. PEA production levels were also enhanced in both aro10 and WT strains transformed with RhPAAS, implying an application for metabolic engineering of PEA biosynthesis in yeast. Characterization of spatial and temporal RhPAAS transcript accumulation in rose revealed it to be specific to floral tissues, peaking in mature flowers, i.e., coinciding with floral scent production and essentially identical to other rose scent-related genes. RhPAAS transcript, as well as PAA and PEA production in flowers, displayed a daily rhythmic behavior, reaching peak levels during the late afternoon hours. Examination of oscillation of RhPAAS transcript levels under free-running conditions suggested involvement of the endogenous clock in the regulation of RhPAAS expression in rose flowers.

Methods for Studying Mitophagy in Yeast.

Under some experimental conditions, eukaryotic cells, from yeast to man, will digest a portion of their mitochondrial cohort through an autophagic process termed mitophagy. In humans, defects in mitophagy have been proposed to play a causative role in a number of late-onset degenerative diseases such as Parkinson's disease and type II diabetes. As a consequence the study of mitophagy, as a quality control process in eukaryotic cells, has become an increasingly important focus in contemporary cell biology. When faced with the task of assaying mitophagy in yeast, the experimentalist has at his or her disposal a variety of induction conditions and assay systems to choose from. Here, we survey several well-established protocols for inducing and monitoring mitophagy in the yeast Saccharomyces cerevisiae and discuss their relative merits, limitations, and potential pitfalls.

Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy.

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.