Hagai Abeliovich

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE‐like product acts in endoplasmic reticulum (ER)‐to‐Golgi transport. To gain insight into the function of Bet3p, we added three c‐myc tags to its C‐terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex (∼800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER‐to‐Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis‐Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.

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 in Yeast: Mechanistic Insights and Physiological Function

SUMMARY Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.

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.

Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE–Sec1p complex composed of Tlg2p and Vps45p

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non‐classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double‐membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single‐membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non‐selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t‐SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45–Tlg2p‐dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t‐SNAREs, but API maturation is not blocked in a pep12ts mutant, indicating that Vps45p independently regulates the function of multiple t‐SNARES at distinct trafficking steps.

Benzoic acid, a weak organic acid food preservative, exerts specific effects on intracellular membrane trafficking pathways in Saccharomyces cerevisiae

ABSTRACT Microbial spoilage of food causes losses of up to 40% of all food grown for human consumption worldwide. Yeast growth is a major factor in the spoilage of foods and beverages that are characterized by a high sugar content, low pH, and low water activity, and it is a significant economic problem. While growth of spoilage yeasts such as Zygosaccharomyces bailii and Saccharomyces cerevisiae can usually be retarded by weak organic acid preservatives, the inhibition often requires levels of preservative that are near or greater than the legal limits. We identified a novel synergistic effect of the chemical preservative benzoic acid and nitrogen starvation: while exposure of S. cerevisiae to either benzoic acid or nitrogen starvation is cytostatic under our conditions, the combination of the two treatments is cytocidal and can therefore be used beneficially in food preservation. In yeast, as in all eukaryotic organisms, survival under nitrogen starvation conditions requires a cellular response called macroautophagy. During macroautophagy, cytosolic material is sequestered by intracellular membranes. This material is then targeted for lysosomal degradation and recycled into molecular building blocks, such as amino acids and nucleotides. Macroautophagy is thought to allow cellular physiology to continue in the absence of external resources. Our analyses of the effects of benzoic acid on intracellular membrane trafficking revealed that there was specific inhibition of macroautophagy. The data suggest that the synergism between nitrogen starvation and benzoic acid is the result of inhibition of macroautophagy by benzoic acid and that a mechanistic understanding of this inhibition should be beneficial in the development of novel food preservation technologies.

Generation of the potent anti-malarial drug artemisinin in tobacco

volume 29 number 12 DeCember 2011 nature biotechnology To the Editor: The emergence of multidrug-resistant strains of Plasmodium spp., the etiological agent of malaria, constitutes a major threat to controlling the disease1,2. Artemisinin, a natural compound from Artemisia annua (sweet wormwood) plants, is highly effective against drug-resistant malaria. Even so, lowcost artemisinin-based drugs are lacking because of the high cost of obtaining natural or chemically synthesized artemisinin1,2. Martin et al.3 were the first to report the generation of an artemisinin precursor in a microbial system. They engineered Escherichia coli with a synthetic mevalonate pathway from Saccharomyces cerevisiae. Expression of amorphadiene synthase (ADS) from A. annua in this strain allowed production of amorpha4,11-diene, the sesquiterpene olefin precursor to artemisinin. However, despite extensive effort invested in the past decade in metabolic engineering of artemisinin and its precursors in both microbial and heterologous plant systems2–6, production of artemisinin itself has never been achieved. Here we report the metabolic engineering of tobacco to produce artemisinin, generating transgenic plants that express five plantand yeast-derived genes involved in the mevalonate and artemisinin pathways, all expressed from a single vector. Our experiments demonstrate that artemisinin can be fully biosynthesized in a heterologous (that is, other than A. annua) plant system, such as tobacco. Although the artemisinin levels we have generated in transgenic tobacco are currently lower than those in A. annua, our experimental platform should lead to the design of new routes for the drug’s commercial production in heterologous plant systems. The World Health Organisation (WHO; Geneva) promotes the use of artemisinin as a first-line treatment for malaria, and it is heavily involved in facilitating the development of artemisinin-based anti-malaria drugs1. Artemisinin is biosynthesized from terpenoid backbones generated by the mevalonate and methyl-erythritol phosphate (MEP) pathways7–9 (Fig. 1a). Although detailed knowledge of the artemisinin-biosynthesis pathway is still lacking, it initiates with the cyclization of farnesyl diphosphate by ADS to form amorpha-4,11-diene, which is then oxidized by the cytochrome P450 CYP71AV1, reduced by artemisinic aldehyde reductase (DBR2) and possibly reoxidized by aldehyde dehydrogenase to yield dihydroartemisinic acid—the presumed precursor of artemisinin in plants6,7,10. Normally, dihydroartemisinic acid accumulates in A. annua and slowly converts to artemisinin, a process that can be stimulated after harvest by drying in the sun. The transformation of dihydroartemisinic acid to artemisinin in A. annua has been proposed to be nonenzymatic, requiring only the presence of light and molecular oxygen6,7; a singlet oxygen formed as a consequence of exposure to UV/visible light may react with dihydroartemisinic acid to form a ketoenol, which can then react with ground-state oxygen to form a second hydroperoxide that spontaneously forms artemisinin. Despite the great interest in enhancing yields of artemisinin in its host A. annua, classic breeding and genetic engineering strategies have met with only limited success6,11. Moreover, recent attempts to produce the artemisinin precursors artemisinic and dihydroartemisinic acids in heterologous plants4,5 did not lead to their accumulation due to internal glycosylation and insufficient oxidation toward the acids. Harnessing E. coli and S. cerevisiae has allowed the production of high titers of amorpha-4,11diene and artemisinic acid2,3, but not of the active artemisinin drug itself. To reconstruct the artemisinin-producing pathway in Nicotiana tabacum, we first generated a mega-vector carrying cytochrome P450 reductase (CPR) from A. annua to prevent the accumulation of inactive oxidized P450, as well as ADS, CYP71AV1 and DBR2 (Fig. 1b and Supplementary Methods). The mega-vector also contained a truncated and deregulated 3-hydroxy-3-methylglutarylcoenzyme A reductase (tHMG) from yeast to increase the supply of precursor from the mevalonate pathway for artemisinin production. To ensure genetic stability and Generation of the potent anti-malarial drug artemisinin in tobacco

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 cells 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 aup1Δ 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.

Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy

ABSTRACT 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.

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

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 genetic 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.