Autophagy induction in atrophic muscle cells requires ULK1 activation by TRIM32 through unanchored K63-linked polyubiquitin chains.

Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32's ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.

Transcriptional and epigenetic profiling of nutrient-deprived cells to identify novel regulators of autophagy.

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway critical for maintaining cellular homeostasis and viability, and is predominantly regarded as a rapid and dynamic cytoplasmic process. To increase our understanding of the transcriptional and epigenetic events associated with autophagy, we performed extensive genome-wide transcriptomic and epigenomic profiling after nutrient deprivation in human autophagy-proficient and autophagy-deficient cells. We observed that nutrient deprivation leads to the transcriptional induction of numerous autophagy-associated genes. These transcriptional changes are reflected at the epigenetic level (H3K4me3, H3K27ac, and H3K56ac) and are independent of autophagic flux. As a proof of principle that this resource can be used to identify novel autophagy regulators, we followed up on one identified target: EGR1 (early growth response 1), which indeed appears to be a central transcriptional regulator of autophagy by affecting autophagy-associated gene expression and autophagic flux. Taken together, these data stress the relevance of transcriptional and epigenetic regulation of autophagy and can be used as a resource to identify (novel) factors involved in autophagy regulation.

Monitoring the Formation of Autophagosomal Precursor Structures in Yeast Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is a powerful and versatile model organism for studying multiple aspects of the biology of eukaryotic cells, including the molecular principles underlying autophagy. One of the unique advantages of this unicellular system is its amenability to genetic and biochemical approaches, which had a pivotal role in the discovery and characterization of most of the autophagy-related (Atg) proteins, the central players of autophagy. The relevance of investigating autophagy in this cell model lies in the high conservation of this pathway among eukaryotes, i.e., most of the yeast Atg proteins possess one or more mammalian orthologs. In addition to the experimental advantages, a very large collection of reagents keeps S. cerevisiae in a leading position for the study of the molecular mechanism and regulation of autophagy. In this chapter, we describe fluorescence microscopy and biochemical methods that allow to monitor in vivo the assembly the of Atg machinery, a key step of autophagy. These approaches can be very useful to those researchers that would like to assess the progression of the autophagosomal precursor structure formation under various conditions, in the presence of specific Atg protein mutants or in the absence of other factors.

Control of Lobesia botrana (Lepidoptera: Tortricidae) by biodegradable ecodian sex pheromone dispensers.

Mating disruption with a high density of sex pheromone dispensers is a new strategy recently developed for the control of the moth Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae). Ecodian LB dispensers, made of low-cost biodegradable material, were formulated with 10 mg of (E,Z) -7,9-dodecadienyl acetate and placed at a rate of 1,600 dispensers per ha. Seasonal dispenser performances were studied using different methods. The female attractiveness disruption and the efficacy of the method were evaluated in the field. The release rates of field-aged Ecodian LB dispensers, measured directly by solid phase microextraction, was comparable with that of the standard monitoring lure after 50-60 d of field exposure and significantly lower beyond 60 d; however, at the end of the season, it was approximately 46 times higher than that of a calling L. botrana female. Electroantennographic recordings showed that dispensers of different field age strongly stimulated male antennae. In a wind tunnel test, dispensers elicited close-range approaches and direct source contacts irrespective of their age. In fields treated with Ecodian dispensers the attractiveness of traps lured with calling females and monitoring baits was significantly reduced. Our data suggest that Ecodian dispensers are active sources of pheromone throughout the season. The efficacy of Ecodian strategy for L. botrana control was comparable with standard mating disruption and curative insecticides.

Control of Cydia pomonella L. and Cydia molesta (Busck) (Lepidoptera Tortricidae) in pome-fruit orchards with Ecodian sex pheromone dispensers.

A mating disruption approach using high densities of pheromone dispensers, has been recently proposed for codling moth, Cydia pomonella (L.), and oriental fruit moth, Cydia molesta (Busck.), (Lepidoptera Tortricidae), control. Ecodian Star dispensers, made of low-cost biodegradable material and easy to apply, were formulated with 10 mg of codlemone (E8,E10-12OH) and 10 mg of grapamone (Z8-12OH) and placed at a rate of 1,400-2,000 dispensers/ha. The pheromone release rates from new and field aged dispensers were evaluated by hexane extraction of the residual attractant (indirectly) and gas-chromatographic analysis. The release rate of field-aged dispensers decreased over time with a good linearity; they released a significant amount of synthetic sex pheromones over the entire season. Dispensers elicited close-range approaches of codling moth males in wind tunnel irrespective of their age. Field trials carried out from 2003 to 2004 confirmed the efficacy of Ecodian Star dispensers for codling moth and oriental fruit moth control, regardless the size of the treated area. Our results demonstrate that Ecodian dispensers achieved a good level of activity and longevity over the season. The potential of this strategy for the control of the moths is discussed.

Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting.

Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle-forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis-sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin-independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.

Polar transmembrane domains target proteins to the interior of the yeast vacuole.

Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly. Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes. However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal. In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum. The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p. It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole. These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.

Deletion of GPI7, a yeast gene required for addition of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity.

Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in Deltagpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9-11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiester-linked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the alpha1,6-linked mannose of the GPI core structure.

Biosynthesis of inositol phosphoceramides and remodeling of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae are mediated by different enzymes.

Metabolic labeling of cells with [3H]dihydrosphingosine ([3H]DHS) allows us to follow the incorporation of this tracer into ceramides (Cer), inositol phosphoceramides (IPCs), and mannosylated IPCs and at the same time to assess the remodeling of glycosylphosphatidylinositol proteins during which preexisting anchor lipid moieties are replaced by [3H]Cer-containing anchors. The results indicate that the remodelases in the endoplasmic reticulum and Golgi use as their substrate Cers that are not generated by the breakdown of IPCs but are newly synthesized. Aureobasidin A, an inhibitor of the IPC synthase Aur1p completely blocks IPC biosynthesis at 0.5 micrograms/ml but does not block remodeling of glycosylphosphatidylinositol anchors even at concentrations up to 10 micrograms/ml. In addition, a synthetic Cer analogue, N-hexanoyl-[3H]DHS, is used as a substrate by Aur1p but not by the remodelases. Thus, remodeling is not mediated by Aur1p although remodeling presumably proceeds by an analogous reaction. Studies with secretion mutants deficient in COPII or COPI coat proteins show that all COPII mutants are unable to introduce [3H]Cer by the Golgi remodelase at the restrictive temperature. This suggests that Cer has to be transported by a COPII-dependent way from the endoplasmic reticulum to Golgi for Golgi remodeling to occur. Golgi remodeling is also not operating in the erd2 mutant and is significantly reduced in COPI mutants, suggesting a dependence of Golgi remodeling on retrotransport.

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid.

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1-6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.

Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids. To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-[2-(3)H]inositol-labelled protein anchors and from GPI intermediates. There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis. However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol. The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1. Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi. In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.

Lipid remodeling leads to the introduction and exchange of defined ceramides on GPI proteins in the ER and Golgi of Saccharomyces cerevisiae.

Previous experiments with Saccharomyces cerevisiae had suggested that diacylglycerol-containing glycosylphosphatidylinositols (GPIs) are added to newly synthesized proteins in the endoplasmic reticulum (ER) and that ceramides subsequently are incorporated into GPI proteins by lipid remodeling. Here we prove this hypothesis by labeling yeast cells with [3H]dihydrosphingosine ([3H]DHS) and showing that this tracer is incorporated into many GPI proteins even when protein synthesis and, hence, anchor addition, is blocked by cycloheximide. [3H]DHS incorporation is greatly enhanced if endogenous synthesis of DHS is inhibited by myriocin. Labeled GPI anchors contain three types of ceramides which, based on previous and present results, are identified as DHS-C26:0, phytosphingosine-C26:0 and phytosphingosine-C26:0-OH, the latter being found only on proteins which have reached the Golgi. Lipid remodeling can occur both in the ER and in a later secretory compartment. In addition, ceramide is incorporated into GPI proteins a long time after their initial synthesis by a process in which one ceramide gets replaced by another ceramide. Remodeling outside the ER requires vesicular flow from the ER to the Golgi, possibly to supply the remodeling enzymes with ceramides.