Identification of a modulator of the actin cytoskeleton, mitochondria, nutrient metabolism and lifespan in yeast.

In yeast, actin cables are F-actin bundles that are essential for cell division through their function as tracks for cargo movement from mother to daughter cell. Actin cables also affect yeast lifespan by promoting transport and inheritance of higher-functioning mitochondria to daughter cells. Here, we report that actin cable stability declines with age. Our genome-wide screen for genes that affect actin cable stability identified the open reading frame YKL075C. Deletion of YKL075C results in increases in actin cable stability and abundance, mitochondrial fitness, and replicative lifespan. Transcriptome analysis revealed a role for YKL075C in regulating branched-chain amino acid (BCAA) metabolism. Consistent with this, modulation of BCAA metabolism or decreasing leucine levels promotes actin cable stability and function in mitochondrial quality control. Our studies support a role for actin stability in yeast lifespan, and demonstrate that this process is controlled by BCAA and a previously uncharacterized ORF YKL075C, which we refer to as actin, aging and nutrient modulator protein 1 (AAN1).

Roles for L o microdomains and ESCRT in ER stress-induced lipid droplet microautophagy in budding yeast.

Microlipophagy (µLP), degradation of lipid droplets (LDs) by microautophagy, occurs by autophagosome-independent direct uptake of LDs at lysosomes/vacuoles in response to nutrient limitations and ER stressors in Saccharomyces cerevisiae. In nutrient-limited yeast, liquid-ordered (Lo) microdomains, sterol-rich raftlike regions in vacuolar membranes, are sites of membrane invagination during LD uptake. The endosome sorting complex required for transport (ESCRT) is required for sterol transport during Lo formation under these conditions. However, ESCRT has been implicated in mediating membrane invagination during µLP induced by ER stressors or the diauxic shift from glycolysis- to respiration-driven growth. Here we report that ER stress induced by lipid imbalance and other stressors induces Lo microdomain formation. This process is ESCRT independent and dependent on Niemann-Pick type C sterol transfer proteins. Inhibition of ESCRT or Lo microdomain formation partially inhibits lipid imbalance-induced µLP, while inhibition of both blocks this µLP. Finally, although the ER stressors dithiothreitol or tunicamycin induce Lo microdomains, µLP in response to these stressors is ESCRT dependent and Lo microdomain independent. Our findings reveal that Lo microdomain formation is a yeast stress response, and stress-induced Lo microdomain formation occurs by stressor-specific mechanisms. Moreover, ESCRT and Lo microdomains play functionally distinct roles in LD uptake during stress-induced µLP.

Membrane dynamics and protein targets of lipid droplet microautophagy during ER stress-induced proteostasis in the budding yeast, Saccharomyces cerevisiae.

Our previous studies reveal a mechanism for lipid droplet (LD)-mediated proteostasis in the endoplasmic reticulum (ER) whereby unfolded proteins that accumulate in the ER in response to lipid imbalance-induced ER stress are removed by LDs and degraded by microlipophagy (µLP), autophagosome-independent LD uptake into the vacuole (the yeast lysosome). Here, we show that dithiothreitol- or tunicamycin-induced ER stress also induces µLP and identify an unexpected role for vacuolar membrane dynamics in this process. All stressors studied induce vacuolar fragmentation prior to µLP. Moreover, during µLP, fragmented vacuoles fuse to form cup-shaped structures that encapsulate and ultimately take up LDs. Our studies also indicate that proteins of the endosome sorting complexes required for transport (ESCRT) are upregulated, required for µLP, and recruited to LDs, vacuolar membranes, and sites of vacuolar membrane scission during µLP. We identify possible target proteins for LD-mediated ER proteostasis. Our live-cell imaging studies reveal that one potential target (Nup159) localizes to punctate structures that colocalizes with LDs 1) during movement from ER membranes to the cytosol, 2) during microautophagic uptake into vacuoles, and 3) within the vacuolar lumen. Finally, we find that mutations that inhibit LD biogenesis, homotypic vacuolar membrane fusion or ESCRT function inhibit stress-induced autophagy of Nup159 and other ER proteins. Thus, we have obtained the first direct evidence that LDs and µLP can mediate ER stress-induced ER proteostasis, and identified direct roles for ESCRT and vacuolar membrane fusion in that process.

Lysosomal recycling of amino acids affects ER quality control.

Recent work has highlighted the fact that lysosomes are a critical signaling hub of metabolic processes, providing fundamental building blocks crucial for anabolic functions. How lysosomal functions affect other cellular compartments is not fully understood. Here, we find that lysosomal recycling of the amino acids lysine and arginine is essential for proper ER quality control through the UPRER. Specifically, loss of the lysine and arginine amino acid transporter LAAT-1 results in increased sensitivity to proteotoxic stress in the ER and decreased animal physiology. We find that these LAAT-1-dependent effects are linked to glycine metabolism and transport and that the loss of function of the glycine transporter SKAT-1 also increases sensitivity to ER stress. Direct lysine and arginine supplementation, or glycine supplementation alone, can ameliorate increased ER stress sensitivity found in laat-1 mutants. These data implicate a crucial role in recycling lysine, arginine, and glycine in communication between the lysosome and ER.

Reciprocal interactions between mtDNA and lifespan control in budding yeast.

Loss of mitochondrial DNA (mtDNA) results in loss of mitochondrial respiratory activity, checkpoint-regulated inhibition of cell cycle progression, defects in growth, and nuclear genome instability. However, after several generations, yeast cells can adapt to the loss of mtDNA. During this adaptation, rho0 cells, which have no mtDNA, exhibit increased growth rates and nuclear genome stabilization. Here, we report that an immediate response to loss of mtDNA is a decrease in replicative lifespan (RLS). Moreover, we find that adapted rho0 cells bypass the mtDNA inheritance checkpoint, exhibit increased mitochondrial function, and undergo an increase in RLS as they adapt to the loss of mtDNA. Transcriptome analysis reveals that metabolic reprogramming to compensate for defects in mitochondrial function is an early event during adaptation and that up-regulation of stress response genes occurs later in the adaptation process. We also find that specific subtelomeric genes are silenced during adaptation to loss of mtDNA. Moreover, we find that deletion of SIR3, a subtelomeric gene silencing protein, inhibits silencing of subtelomeric genes associated with adaptation to loss of mtDNA, as well as adaptation-associated increases in mitochondrial function and RLS extension.

Lipid droplet autophagy during energy mobilization, lipid homeostasis and protein quality control.

Lipid droplets (LDs) have well-established functions as sites for lipid storage and energy mobilization to meet the metabolic demands of cells. However, recent studies have expanded the roles of LDs to protein quality control. Lipophagy, or LD degradation by autophagy, plays a vital role not only in the mobilization of free fatty acids (FFAs) and lipid homeostasis at LDs, but also in the adaptation of cells to certain forms of stress including lipid imbalance. Recent studies have provided new mechanistic insights about the diverse types of lipophagy, in particular microlipophagy. This review summarizes key findings about the mechanisms and functions of lipophagy and highlights a novel function of LD microlipophagy as a mechanism to maintain endoplasmic reticulum (ER) proteostasis under conditions of lipid imbalance.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.

Role for Lipid Droplet Biogenesis and Microlipophagy in Adaptation to Lipid Imbalance in Yeast.

The immediate responses to inhibition of phosphatidylcholine (PC) biosynthesis in yeast are altered phospholipid levels, slow growth, and defects in the morphology and localization of ER and mitochondria. With chronic lipid imbalance, yeast adapt. Lipid droplet (LD) biogenesis and conversion of phospholipids to triacylglycerol are required for restoring some phospholipids to near-wild-type levels. We confirmed that the unfolded protein response is activated by this lipid stress and find that Hsp104p is recruited to ER aggregates. We also find that LDs form at ER aggregates, contain polyubiquitinated proteins and an ER chaperone, and are degraded in the vacuole by a process resembling microautophagy. This process, microlipophagy, is required for restoration of organelle morphology and cell growth during adaptation to lipid stress. Microlipophagy does not require ATG7 but does requires ESCRT components and a newly identified class E VPS protein that localizes to ER and is upregulated by lipid imbalance.

Fabrication and characterization of ultrahigh-volume- fraction aligned carbon nanotube-polymer composites.

Aligned CNT nanocomposites with variable volume fraction, up to 20%, are demonstrated. Biaxial mechanical densification of aligned CNT forests, followed by capillarity-driven wetting using unmodified aerospace-grade polymers, creates centimeter-scale specimens. Characterizations confirm CNT alignment and dispersion in the thermosets, providing a useful platform for controlled nanoscale interaction and nanocomposite property studies that emphasize anisotropy.