LDO proteins and Vac8 form a vacuole-lipid droplet contact site to enable starvation-induced lipophagy in yeast.

Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy.

Characterization of the Multiple Domains of Pex30 Involved in Subcellular Localization of the Protein and Regulation of Peroxisome Number.

Pex30 is a peroxisomal protein whose role in peroxisome biogenesis via the endoplasmic reticulum has been established. It is a 58 KDa multi-domain protein that facilitates contact site formation between various organelles. The present study aimed to investigate the role of various domains of the protein in its sub-cellular localization and regulation of peroxisome number. For this, we created six truncations of the protein (1-87, 1-250, 1-352, 88-523, 251-523 and 353-523) and tagged GFP at the C-terminus. Biochemical methods and fluorescence microscopy were used to characterize the effect of truncation on expression and localization of the protein. Quantitative analysis was performed to determine the effect of truncation on peroxisome number in these cells. Expression of the truncated variants in cells lacking PEX30 did not cause any effect on cell growth. Interestingly, variable expression and localization of the truncated variants in both peroxisome-inducing and non-inducing medium was observed. Truncated variants depicted different distribution patterns such as punctate, reticulate and cytosolic fluorescence. Interestingly, lack of the complete dysferlin domain or C-Dysf resulted in increased peroxisome number similar to as reported for cells lacking Pex30. No contribution of this domain in the reticulate distribution of the proteins was also observed. Our results show an interesting role for the various domains of Pex30 in localization and regulation of peroxisome number.

Pex30 undergoes phosphorylation and regulates peroxisome number in Saccharomyces cerevisiae.

Pex30 is a dysferlin domain-containing protein whose role in peroxisome biogenesis has been studied by several research groups. Notably, recent studies have linked this protein to peroxisomes, endoplasmic reticulum and lipid bodies in Saccharomyces cerevisiae. Phosphoproteome studies of S. cerevisiae have identified several phosphorylation sites in Pex30. In this study we expressed and purified Pex30 from its native host. Analysis of the purified protein by circular dichroism spectroscopy showed that it retained its secondary structure and revealed primarily a helical structure. Further phosphorylation of Pex30 at three residues, Threonine 60, Serine 61 and Serine 511 was identified by mass spectrometry in this study. To understand the importance of this post-translational modification in peroxisome biogenesis, the identified residues were mutated to both non-phosphorylatable (alanine) and phosphomimetic (aspartic acid) variants. Upon analysis of the mutant variants by fluorescence microscopy, no alteration in the localization of the protein to ER and peroxisomes was observed. Interestingly, reduced number of peroxisomes were observed in cells expressing phosphomimetic mutations when cultured in peroxisome-inducing conditions. Our data suggest that phosphorylation and dephosphorylation of Pex30 may promote distinct interactions essential in regulating peroxisome number in a cell.

Post-translational modifications of proteins associated with yeast peroxisome membrane: An essential mode of regulatory mechanism.

Peroxisomes are single membrane-bound organelles important for the optimum functioning of eukaryotic cells. Seminal discoveries in the field of peroxisomes are made using yeast as a model. Several proteins required for the biogenesis and function of peroxisomes are identified to date. As with proteins involved in other major cellular pathways, peroxisomal proteins are also subjected to regulatory post-translational modifications. Identification, characterization and mapping of these modifications to specific amino acid residues on proteins are critical toward understanding their functional significance. Several studies have tried to identify post-translational modifications of peroxisomal proteins and determine their impact on peroxisome structure and function. In this manuscript, we provide an overview of the various post-translational modifications that govern the peroxisome dynamics in yeast.