Publications by authors named "Hagai Abeliovich"

38 Publications

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

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

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 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.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

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

Authors:
Hagai Abeliovich

J Biol Chem 2019 11;294(46):17186-17187

Institute of Biochemistry, Food Science, and Nutrition, Hebrew University of Jerusalem, Rehovot 76100, Israel

NSF (-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 , 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 now reports a small molecule that appears to selectively inhibit Sec18 action in an assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming.
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http://dx.doi.org/10.1074/jbc.H119.011334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873167PMC
November 2019

Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20517-20527. Epub 2019 Sep 23.

Institute of Biochemistry, Food Science and Nutrition, Hebrew University of Jerusalem, 7612001 Rehovot, Israel;

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.
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http://dx.doi.org/10.1073/pnas.1901759116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789754PMC
October 2019

Methods for Studying Mitophagy in Yeast.

Methods Mol Biol 2019 ;1880:669-678

Biochemistry, Food Science and Nutrition, Hebrew University of Jerusalem, Rehovot, Israel.

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.
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http://dx.doi.org/10.1007/978-1-4939-8873-0_44DOI Listing
June 2019

Selective emodin toxicity in cancer cells.

Oncotarget 2017 Jun;8(23):36932-36933

Department of Biochemistry and Food Science, Hebrew University of Jerusalem, Rehovot, Israel.

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http://dx.doi.org/10.18632/oncotarget.16611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514881PMC
June 2017

Cardiolipin Regulates Mitophagy through the Protein Kinase C Pathway.

J Biol Chem 2017 02 5;292(7):2916-2923. Epub 2017 Jan 5.

From the Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202 and

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 , , and synthetically interact with CL synthase mutant Δ. 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 , , , and exacerbated Δ 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 Δ cells, and up-regulation of the PKC pathway by expression of the gene, which encodes constitutively activated Pkc1p, rescued Δ 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.
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http://dx.doi.org/10.1074/jbc.M116.753574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314186PMC
February 2017

Roles of mitophagy in cellular physiology and development.

Cell Tissue Res 2017 Jan 3;367(1):95-109. Epub 2016 Aug 3.

Department of Biochemistry and Food Science, Hebrew University of Jerusalem, Rehovot, 76100, Israel.

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.
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http://dx.doi.org/10.1007/s00441-016-2472-0DOI Listing
January 2017

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

Biochem Soc Trans 2016 Apr;44(2):541-5

Department of Dermatology, Medical Center, Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany ZBSA Center for Biological Systems Analysis, University of Freiburg, Habsburgerstr. 49, 79104 Freiburg, Germany Department of Biology, University of Fribourg, Chemin du Musée 10, 1700 Fribourg, Switzerland.

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.
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http://dx.doi.org/10.1042/BST20150278DOI Listing
April 2016

On Hill coefficients and subunit interaction energies.

Authors:
Hagai Abeliovich

J Math Biol 2016 12 1;73(6-7):1399-1411. Epub 2016 Apr 1.

Department of Biochemistry and Food Science, Hebrew University of Jerusalem, 76100, Rehovot, Israel.

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.
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http://dx.doi.org/10.1007/s00285-016-1001-9DOI Listing
December 2016

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

Appl Microbiol Biotechnol 2015 Oct 9;99(20):8667-80. Epub 2015 Jun 9.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.

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.
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http://dx.doi.org/10.1007/s00253-015-6708-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497686PMC
October 2015

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

Authors:
Hagai Abeliovich

Amino Acids 2015 Oct 29;47(10):2165-75. Epub 2014 Jun 29.

Department of Biochemistry, Food Science and Nutrition, Hebrew University of Jerusalem, Rehovot, Israel.

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.
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http://dx.doi.org/10.1007/s00726-014-1787-yDOI Listing
October 2015

Musical chairs during mitophagy.

Autophagy 2014 Apr 12;10(4):706-7. Epub 2014 Feb 12.

Institute of Biochemistry, Food Science and Nutrition; R.H. Smith Faculty of Agriculture, Food and Environment; Hebrew University of Jerusalem; Rehovot, Israel.

Mitophagy, or the autophagic degradation of mitochondria, is thought to be important in mitochondrial quality control, and hence in cellular physiology. Defects in mitophagy correlate with late onset pathologies and aging. Here, we discuss recent results that shed light on the interrelationship between mitophagy and mitochondrial dynamics, based on proteomic analyses of protein dynamics in wild-type and mutant cells. These studies show that different mitochondrial matrix proteins undergo mitophagy at different rates, and that the rate differences are affected by mitochondrial dynamics. These results are consistent with models in which phase separation within the mitochondrial matrix leads to unequal segregation of proteins during mitochondrial fission. Repeated fusion and fission cycles may thus lead to "distillation" of components that are destined for degradation.
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http://dx.doi.org/10.4161/auto.28150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091161PMC
April 2014

Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy.

Nat Commun 2013 ;4:2789

1] The Institute for Biochemistry, Food Science, and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, Israel 76100 [2] Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center Building 35, Room 2C-917 35 Convent Drive, Bethesda, Maryland 20892-3704, USA.

Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells, and defects in mitophagy correlate with ageing phenomena and with several neurodegenerative disorders. A central mechanistic question regarding mitophagy is whether mitochondria are consumed en masse, or whether an active process segregates defective molecules from functional ones within the mitochondrial network, thus allowing a more efficient culling mechanism. Here we combine a proteomic study with a molecular genetics and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We find that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism coupling weak physical segregation with mitochondrial dynamics to achieve a distillation-like effect. In agreement, the rates of mitophagic degradation strongly correlate with the degree of physical segregation of specific matrix proteins.
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http://dx.doi.org/10.1038/ncomms3789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909740PMC
July 2014

Autophagy researchers.

Autophagy 2013 Nov 20;9(11):1648-52. Epub 2013 Sep 20.

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http://dx.doi.org/10.4161/auto.26359DOI Listing
November 2013

Role of membrane association and Atg14-dependent phosphorylation in beclin-1-mediated autophagy.

Mol Cell Biol 2013 Sep 22;33(18):3675-88. Epub 2013 Jul 22.

Biochemistry Section, National Institute for Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.

During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.
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http://dx.doi.org/10.1128/MCB.00079-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753860PMC
September 2013

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

Appl Environ Microbiol 2013 Jan 9;79(2):728-33. Epub 2012 Nov 9.

Department of Food Science and Technology, Oregon State University, Corvallis, Oregon, USA.

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.
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http://dx.doi.org/10.1128/AEM.01737-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553748PMC
January 2013

Guidelines for the use and interpretation of assays for monitoring autophagy.

Autophagy 2012 Apr;8(4):445-544

Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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http://dx.doi.org/10.4161/auto.19496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883PMC
April 2012

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

Plant J 2012 Jan 17;69(1):57-69. Epub 2011 Oct 17.

Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel.

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.
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http://dx.doi.org/10.1111/j.1365-313X.2011.04770.xDOI Listing
January 2012

Harnessing yeast subcellular compartments for the production of plant terpenoids.

Metab Eng 2011 Sep 18;13(5):474-81. Epub 2011 May 18.

Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel.

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.
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http://dx.doi.org/10.1016/j.ymben.2011.05.001DOI Listing
September 2011

Stationary-phase mitophagy in respiring Saccharomyces cerevisiae.

Authors:
Hagai Abeliovich

Antioxid Redox Signal 2011 May 8;14(10):2003-11. Epub 2011 Mar 8.

Department of Biochemistry and Food Science, Hebrew University of Jerusalem, Rehovot, Israel.

The clearance of malfunctioning mitochondria is an important housekeeping function in respiring eukaryotic cells and plays a role in physiological homeostasis as well as in the progression of late-onset diseases. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy. Although the mechanism of nonspecific macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. An important step toward elucidating the mechanism by which defective mitochondria are selected and degraded is the establishment of conditions under which mitophagy is induced. This review covers our current understanding of mitophagy in the model organism Saccharomyces cerevisiae and its modes of activation, with a focus on stationary-phase mitophagy-a form of mitophagy that holds promise as a potential quality control mechanism.
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http://dx.doi.org/10.1089/ars.2010.3807DOI Listing
May 2011

Genetics of flavonoid, carotenoid, and chlorophyll pigments in melon fruit rinds.

J Agric Food Chem 2010 Oct;58(19):10722-8

Newe Yaar Research Center, ARO, PO Box 1021, Ramat Yishay 30095, Israel.

External color has profound effects on acceptability of agricultural products by consumers. Carotenoids and chlorophylls are known to be the major pigments of melon (Cucumis melo L.) rinds. Flavonoids (especially chalcones and anthocyanins) are also prominent in other fruits but have not been reported to occur in melons fruit. We analyzed the pigments accumulating in rinds of different melon genotypes during fruit development. We found that melon rind color is based on different combinations of chlorophyll, carotenoids, and flavonoids according to the cultivar tested and their ratios changed during fruit maturation. Moreover, in "canary yellow" type melons, naringenin chalcone, a yellow flavonoid pigment previously unknown to occur in melons, has been identified as the major fruit colorant in mature rinds. Naringenin chalcone is also prominent in other melon types, occurring together with carotenoids (mainly β-carotene) and chlorophyll. Both chlorophyll and carotenoid pigments segregate jointly in an F(2) population originating from a cross between a yellow canary line and a line with green rind. In contrast, the content of naringenin chalcone segregates as a monogenic trait independently to carotenoids and chlorophyll. Transcription patterns of key structural phenylpropanoid and flavonoid biosynthetic pathway genes were monitored in attempts to explain naringenin chalcone accumulation in melon rinds. The transcript levels of CHI were low in both parental lines, but C4H, C4L, and CHS transcripts were upregulated in "Noy Amid", the parental line that accumulates naringenin chalcone. Our results indicate that naringenin chalcone accumulates independently from carotenoids and chlorophyll pigments in melon rinds and gives an insight into the molecular mechanism for the accumulation of naringenin chalcone in melon rinds.
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http://dx.doi.org/10.1021/jf1021797DOI Listing
October 2010

Induction of autophagic flux by amino acid deprivation is distinct from nitrogen starvation-induced macroautophagy.

Autophagy 2010 Oct 22;6(7):879-90. Epub 2010 Oct 22.

Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot, Israel.

A number of signaling mechanisms have been implicated in the regulation of autophagic trafficking. TOR kinase activity, cAMP levels, and the GAAC pathway have all been suggested to be involved. Here, we closely analyzed the stimuli that underlie induction of autophagic trafficking in Saccharomyces cerevisiae. We find evidence for the existence of a novel aspect of the autophagic pathway that is regulated by intracellular amino acids, uncoupled from extracellular nutrient levels, and is absolutely dependent on Gcn2 and Gcn4. This requirement for Gcn2 and Gcn4 distinguishes amino-acid starvation induced autophagy from classic macroautophagy: Macroautophagic flux in response to nitrogen starvation is only partly diminished in gcn2Δ and gcn4Δ cells. However this maintenance of autophagic flux in gcn mutants during nitrogen starvation reflects the formation of larger numbers of smaller autophagosomes. We report that gcn2Δ and gcn4Δ cells are defective in the induction of Atg8 and Atg4 upon starvation, and this defect results, during total nitrogen starvation, in the formation of abnormally small autophagosomes, although overall autophagic flux remains close to normal due to a compensatory increase in the overall number of autophagosomes.
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http://dx.doi.org/10.4161/auto.6.7.12753DOI Listing
October 2010

Identification of rose phenylacetaldehyde synthase by functional complementation in yeast.

Plant Mol Biol 2010 Feb 1;72(3):235-45. Epub 2009 Nov 1.

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, 76100, Rehovot, Israel.

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.
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http://dx.doi.org/10.1007/s11103-009-9564-0DOI Listing
February 2010

Aup1-mediated regulation of Rtg3 during mitophagy.

J Biol Chem 2009 Dec;284(51):35885-95

Department of Biochemistry and Food Science, Faculty of Agricultural, Food, and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel.

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.
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http://dx.doi.org/10.1074/jbc.M109.048140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2791017PMC
December 2009

Autophagy in food biotechnology.

Autophagy 2009 Oct 5;5(7):925-9. Epub 2009 Oct 5.

Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel.

The purpose of this review is not to explain autophagy (as clearly there is a plethora of reviews and research papers on the topic) but to provide the autophagy-savvy reader with an overview of the impact of autophagy research on a number of current topics in food biotechnology. To understand this connection, we need to remember that autophagy is, at the end of the day, a type of stress response. Since as humans we are heterotrophic eukaryotic organisms, our cells, and the cells of those organisms that we consume, use autophagy as part of the day-to-day business of living. Thus, a number of food biotechnology processes such as brewing and winemaking employ eukaryotic organisms under autophagy-inducing conditions, as noted below. In addition, food spoilage processes also involve eukaryotic organisms and these processes also involve physiological aspects that impinge on autophagy. Finally, the recently introduced concept of "functional foods" introduces the possibility of engineering foodstuff for the induction or inhibition of autophagy in the consumer, with a potential promise of health benefits that merits further research. In this review, we will provide a perspective on the current literature in these three areas, their relationship to current basic research in autophagy, and their future applicative potential.
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http://dx.doi.org/10.4161/auto.5.7.9213DOI Listing
October 2009

Monitoring autophagy in yeast using FM 4-64 fluorescence.

Methods Enzymol 2008 ;451:79-88

Department of Biochemistry and Food Science, Hebrew University of Jerusalem, Rehovot, Israel.

The original observations and experiments dealing with autophagy were purely morphological in nature. Even though more and more molecular techniques have been introduced, experimenters are often asked to provide visual evidence of autophagic processes in order to back up data obtained via other means. In yeast as well, autophagosomes were initially defined morphologically and indirectly, by observing intravacuolar autophagic bodies that accumulate upon starvation. This can be achieved by electron microscopy, which affords very high resolution but is time consuming and costly, or by light microscopy, which is a relatively inaccurate method of scoring autophagy. A third alternative, which we present here, is to use the unique properties of the fluorescent dye FM 4-64 to follow the accumulation of autophagic bodies.
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http://dx.doi.org/10.1016/S0076-6879(08)03207-2DOI Listing
March 2009

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Autophagy 2008 Feb 21;4(2):151-75. Epub 2007 Nov 21.

Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654259PMC
http://dx.doi.org/10.4161/auto.5338DOI Listing
February 2008

Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid.

Autophagy 2008 Jan 8;4(1):28-36. Epub 2007 Oct 8.

Department of Biochemistry and Food Science, Hebrew University of Jerusalem, Rehovot, Israel.

Weak organic acids are an important class of food preservatives that are particularly efficacious towards yeast and fungal spoilage. While acids with small aliphatic chains appear to function by acidification of the cytosol and are required at high concentrations to inhibit growth, more hydrophobic organic acids such as sorbic and benzoic acid have been suggested to function by perturbing membrane dynamics and are growth-inhibitory at much lower concentrations. We previously demonstrated that benzoic acid has selective effects on membrane trafficking in Saccharomyces cerevisiae. Benzoic acid selectively blocks macroautophagy in S. cerevisiae while acetic acid does not, and sorbic acid does so to a lesser extent. Indeed, while both benzoic acid and nitrogen starvation are cytostatic when assayed separately, the combination of these treatments is cytocidal, because macroautophagy is essential for survival during nitrogen starvation. In this report, we demonstrate that Zygosaccharomyces bailii, a food spoilage yeast with relatively high resistance to weak acid stress, also exhibits a cytocidal response to the combination of benzoic acid and nitrogen starvation. In addition, we show that nitrogen starvation can be replaced by caffeine supplementation. Caffeine induces a starvation response that includes the induction of macroautophagy, and the combination of caffeine and benzoic acid is cytocidal, as predicted from the nitrogen starvation data.
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http://dx.doi.org/10.4161/auto.5127DOI Listing
January 2008
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