Eduardo Cebollero

Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories

During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

Regulation of autophagy in yeast Saccharomyces cerevisiae

Autophagy is a conserved catabolic process that initially involves the bulk or the selective engulfment of cytosolic components into double-membrane vesicles and successively the transport of the sequestered cargo material into the lysosome/vacuole for degradation. This pathway allows counteracting internal and external stresses, including changes in the nutrient availability, that alter the cell metabolic equilibrium. Consequently, the regulation of autophagy is crucial for maintaining important cellular functions under various conditions and ultimately it is essential for survival. Yeast Saccharomyces cerevisiae has been successfully employed as a model system to study autophagy. For instance, it has allowed the isolation of the factors specifically involved in autophagy, the Atg proteins, and the characterization of some of their molecular roles. In addition, this organism also possesses all the principal signaling cascades that modulate the cell metabolism in response to nutrient availability in higher eukaryotes, including the TOR and the PKA pathways. Therefore, yeast is an ideal system to study the regulation of autophagy by these signaling pathways. Here, we review the current state of our knowledge about the molecular events leading to the induction or inhibition of autophagy in yeast with special emphasis on the regulation of the function of Atg proteins.

Phosphatidylinositol-3-phosphate clearance plays a key role in autophagosome completion

BACKGROUND The biogenesis of autophagosomes, the hallmark of autophagy, depends on the function of the autophagy-related (Atg) proteins and the generation of phosphatidylinositol-3-phosphate (PtdIns3P) at the phagophore assembly site (PAS), the location where autophagosomes arise. The current model is that PtdIns3P is involved primarily in the recruitment of Atg proteins to the PAS and that once an autophagosome is complete, the Atg machinery is released from its surface back into the cytoplasm and reused for the formation of new vesicles. RESULTS We have identified a PtdIns3P phosphatase, Ymr1, that is essential for the normal progression of both bulk and selective types of autophagy. This protein is recruited to the PAS at an early stage of formation of this structure through a process that requires both its GRAM domain and its catalytic activity. In the absence of Ymr1, Atg proteins fail to dissociate from the limiting membrane of autophagosomes, and these vesicles accumulate in the cytoplasm. CONCLUSIONS Our data thus reveal a key role for PtdIns3P turnover in the regulation of the late steps of autophagosome biogenesis and indicate that the disassembly of the Atg machinery from the surface of autophagosomes is a requisite for their fusion with the vacuole.

A Recombinant Saccharomyces cerevisiae Strain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

ABSTRACT Stabilization against protein haze was one of the first positive properties attributed to yeast mannoproteins in winemaking. In previous work we demonstrated that deletion of KNR4 leads to increased mannoprotein release in laboratory Saccharomyces cerevisiae strains. We have now constructed strains with KNR4 deleted in two different industrial wine yeast backgrounds. This required replacement of two and three alleles of KNR4 for the EC1118 and T73-4 backgrounds, respectively, and the use of three different selection markers for yeast genetic transformation. The actual effect of the genetic modification was dependent on both the genetic background and the culture conditions. The fermentation performance of T73-4 derivatives was clearly impaired, and these derivatives did not contribute to the protein stability of the wine, even though they showed increased mannoprotein release in vitro. In contrast, the EC1118 derivative with both alleles of KNR4 deleted released increased amounts of mannoproteins both in vitro and during wine fermentation assays, and the resulting wines were consistently less susceptible to protein haze. The fermentation performance of this strain was slightly impaired, but only with must with a very high sugar content. These results pave the way for the development of new commercial strains with the potential to improve several mannoprotein-related quality and technological parameters of wine.

Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

ABSTRACT Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.

Atg18 function in autophagy is regulated by specific sites within its β-propeller

Summary Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a &bgr;-propeller, formed by seven WD40 repeats, that contains a conserved FRRG motif that binds to phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. However, it is unknown how Atg18 association with these organelles is regulated, as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds to Atg2 through a specific stretch of amino acids in the &bgr;-propeller on the opposite surface to the FRRG motif. As in the absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model whereby the Atg18 &bgr;-propeller provides organelle specificity by binding to two determinants on the target membrane.

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.

Evidence for Yeast Autophagy during Simulation of Sparkling Wine Aging: A Reappraisal of the Mechanism of Yeast Autolysis in Wine

Yeast autolysis is the source of several molecules responsible for the quality of wines aged in contact with yeast cells. However, the mechanisms of yeast autolysis during wine aging are not completely understood. All descriptions of yeast autolysis in enological conditions emphasize the disturbance of cell organization as the starting event in the internal digestion of the cell, while no reference to autophagy is found in wine‐related literature. By using yeast mutants defective in the autophagic or the Cvt pathways we have demonstrated that autophagy does take place in wine production conditions. This finding has implications for the genetic improvement of yeasts for accelerated autolysis.

Mitochondria: One of the origins for autophagosomal membranes?

Macroautophagy is a transport pathway to the lysosome/vacuole that contributes to the degradation of numerous intracellular components. Despite the recent advances achieved in the understanding of the molecular mechanism underlying macroautophagy, the membrane origin of autophagosomes, the hallmark of this process is still a mystery. It has been suggested that mitochondria may be one of the lipid sources for autophagosome formation and that possibly this organelle provides the phosphatidylethanolamine (PE) that covalently links to the members of the ubiquitin-like Atg8/microtubule-associated protein 1 light chain 3 (LC3) protein family. These lipidated proteins are inserted into the outer and inner surface of autophagosomes and are essential for the biogenesis of these large double-membrane vesicles. However, because PE is an integral component of all cellular membranes, designing appropriate experiments to determine the origin of the autophagosomal PE is not easy. In this review, we discuss the idea that mitochondria provide the pool of PE necessary for the autophagosome biogenesis and we propose some possible experimental approaches aimed to explore this possibility.

Multilocus sequence typing of oenological Saccharomyces cerevisiae strains

This study describes the application of a multilocus sequence typing (MLST) analysis for molecular discrimination at the strain level of Spanish wine yeast strains. The discrimination power of MLST is compared to mitochondrial RFLP analysis. Fragments of the ADP1, ACC1, RPN2, GLN4, and ALA1 genes were amplified by PCR from chromosomal DNA of 18 wine Saccharomyces cerevisiae strains. Ten polymorphic sites were found in the five loci analyzed showing 13 different genotypes, with 11 of them represented by only one strain. RFLP analysis of the same 18 wine yeast strains showed seventeen different mitochondrial patterns. Phylogenetic relationships among the strains analyzed, inferred by MLST data, showed wine isolates of S. cerevisiae as a rather homogeneous group. The discrimination potential of mitochondrial RFLP analysis was superior to the MLST scheme used in this work. However, MLST analysis allowed an easy construction of reliable phylogenetic trees. MLST analysis offers the possibility of typing wine S. cerevisiae strains simultaneously to the study of the genetic relationship among them.