Autophagy is a major catabolic process responsible for the delivery of proteins and organelles to the lysosome/vacuole for degradation. Malfunction of this pathway has been implicated in numerous pathological conditions. Different organelles have been found to contribute to the formation of autophagosomes, but the exact mechanism mediating this process remains obscure. Here, we show that lipid droplets (LDs) are important for the regulation of starvation-induced autophagy. Deletion of Dga1 and Lro1 enzymes responsible for triacylglycerol (TAG) synthesis, or of Are1 and Are2 enzymes responsible for the synthesis of steryl esters (STE), results in the inhibition of autophagy. Moreover, we identified the STE hydrolase Yeh1 and the TAG lipase Ayr1 as well as the lipase/ hydrolase Ldh1 as essential for autophagy. Finally, we provide evidence that the ER-LD contact-site proteins Ice2 and Ldb16 regulate autophagy. Our study thus highlights the importance of lipid droplet dynamics for the autophagic process under nitrogen starvation.

Cdc48/p97/VCP plays a ubiquitin-independent role during autophagosome formation in S. cerevisiae.

Rapid estimation of the macroautophagi crate has become of great importance over the past few years. A variety of methods to follow autophagy were established both in S. cerevisiae and the mammalian system. In yeast,measuring the breakdown of GFP-Atg8,and in mammalian cells counting the increase of LC3 puncta, have become the most commonly used assays to quantify autophagy. Here, we provide degradation of Pgk1-GFP followed in immunoblots as a new convenient tool to quantify nonselective bulk autophagy in yeast.

Autophagy, an evolutionarily conserved intracellular catabolic process, leads to the degradation of cytosolic proteins and organelles in the vacuole/lysosome. Different forms of selective autophagy have recently been described. Starvation-induced protein degradation, however, is considered to be nonselective. Here we describe a novel interaction between autophagy-related protein 8 (Atg8) and fatty acid synthase (FAS), a pivotal enzymatic complex responsible for the entire synthesis of C16-and C18-fatty acids in yeast. We show that although FAS possesses housekeeping functions, under starvation conditions it is delivered to the vacuole for degradation by autophagy in a Vac8-and Atg24-dependent manner. We also provide evidence that FAS degradation is essential for survival under nitrogen deprivation. Our results imply that during nitrogen starvation specific proteins are preferentially recruited into autophagosomesA utophagy is a major cellular catabolic pathway in eukaryotes, responsible for the degradation of organelles and large protein aggregates. This process is induced under various stress conditions, such as amino acid starvation, hypoxia, and oxidative stress (1, 2).In yeast (Saccharomyces cerevisiae), nitrogen starvation is known to induce macroautophagy (hereafter termed autophagy), which is essential mainly for supplementing amino acids needed for protein synthesis (3). Autophagy has long been considered to be a nonselective process except for the recruitment of ribosomes into autophagosomes, which was suggested to be selective under conditions of nitrogen starvation (4). Selective autophagy is thought to play a role mainly in cell homeostasis, and it uses the core autophagy genes and varying sets of proteins for different cargos (5). Autophagy-related protein 8 (Atg8), a key autophagic factor essential for autophagosome biogenesis, also plays a major role in selective autophagy by recruiting cargo proteins into the autophagosome (6).Fatty acid synthase (FAS) is a large (2.6 MDa), essential enzymatic complex responsible for the entire synthesis of C16-and C18-fatty acids (7). FAS is composed of two subunits, Fas1 (Fasβ) and Fas2 (Fasα), which are arranged in an α 6 β 6 macromolecular complex. Deletion of one of the FAS subunits has been shown to result in degradation of the other (8). Whereas the unassembled Fas2 subunit is short-lived and degrades in the proteasome, the unassembled Fas1, once induced, is degraded by autophagy. Moreover, the FAS complex is known to be delivered to the vacuole under potassium acetate starvation (8).Here we investigated the role of autophagy in the degradation of the FAS complex. We report a physical interaction between Atg8 and FAS, and show that autophagy preferentially degrades FAS and decreases its activity during nitrogen starvation. We suggest that the selective degradation of FAS under nitrogen starvation requires the participation of Atg24 and Vac8, two factors that each play a role in selective autophagy (9-12). Finally, we show that FAS degradation is important ...

Mitochondria are turned over by an autophagic process termed mitophagy. This process is considered to remove damaged, superfluous and aged organelles. However, little is known about how defective organelles are recognized, what types of damage induce turnover, and whether an identical set of factors contributes to degradation under different conditions. Here we systematically compared the mitophagy rate and requirement for mitophagy-specific proteins during post-log-phase and rapamycin-induced mitophagy. To specifically assess mitophagy of damaged mitochondria, we analyzed cells accumulating proteins prone to degradation due to lack of the mitochondrial AAA-protease Yme1. While autophagy 32 (Atg32) was required under all tested conditions, the function of Atg33 could be partially bypassed in post-log-phase and rapamycin-induced mitophagy. Unexpectedly, we found that Uth1 was dispensable for mitophagy. A re-evaluation of its mitochondrial localization revealed that Uth1 is a protein of the inner mitochondrial membrane that is targeted by a cleavable N-terminal pre-sequence. In agreement with our functional analyses, this finding excludes a role of Uth1 as a mitochondrial surface receptor.