Aneuploidy-induced proteotoxic stress can be effectively tolerated without dosage compensation, genetic mutations, or stress responses.

BACKGROUND: The protein homeostasis (proteostasis) network maintains balanced protein synthesis, folding, transport, and degradation within a cell. Failure to maintain proteostasis is associated with aging and disease, leading to concerted efforts to study how the network responds to various proteotoxic stresses. This is often accomplished using ectopic overexpression of well-characterized, model misfolded protein substrates. However, how cells tolerate large-scale, diverse burden to the proteostasis network is not understood. Aneuploidy, the state of imbalanced chromosome content, adversely affects the proteostasis network by dysregulating the expression of hundreds of proteins simultaneously. Using aneuploid haploid yeast cells as a model, we address whether cells can tolerate large-scale, diverse challenges to the proteostasis network. RESULTS: Here we characterize several aneuploid Saccharomyces cerevisiae strains isolated from a collection of stable, randomly generated yeast aneuploid cells. These strains exhibit robust growth and resistance to multiple drugs which induce various forms of proteotoxic stress. Whole genome re-sequencing of the strains revealed this was not the result of genetic mutations, and transcriptome profiling combined with ribosome footprinting showed that genes are expressed and translated in accordance to chromosome copy number. In some strains, various facets of the proteostasis network are mildly upregulated without chronic activation of environmental stress response or heat shock response pathways. No severe defects were observed in the degradation of misfolded proteins, using model misfolded substrates of endoplasmic reticulum-associated degradation or cytosolic quality control pathways, and protein biosynthesis capacity was not impaired. CONCLUSIONS: We show that yeast strains of some karyotypes in the genetic background studied here can tolerate the large aneuploidy-associated burden to the proteostasis machinery without genetic changes, dosage compensation, or activation of canonical stress response pathways. We suggest that proteotoxic stress, while common, is not always an obligate consequence of aneuploidy, but rather certain karyotypes and genetic backgrounds may be able to tolerate the excess protein burden placed on the protein homeostasis machinery. This may help clarify how cancer cells are paradoxically both highly aneuploid and highly proliferative at the same time.

Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated.

Inositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidase YPC1 rescues cells under this condition. We established hydroxylated C26 fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only when YPC1 is overexpressed. In contrast, overexpression of YPC1 has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidant N-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.

Characterization of yeast mutants lacking alkaline ceramidases YPC1 and YDC1.

Humans and yeast possess alkaline ceramidases located in the early secretory pathway. Single deletions of the highly homologous yeast alkaline ceramidases YPC1 and YDC1 have very little genetic interactions or phenotypes. Here, we performed chemical-genetic screens to find deletions/conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities in sphingolipid biosynthesis detected by metabolic labeling do not alter the mass spectrometric lipid profile of ypc1∆ydc1∆ cells. Ceramides of ypc1∆ydc1∆ remained normal even in presence of aureobasidin A, an inhibitor of inositolphosphorylceramide synthase. Moreover, in caloric restriction conditions Ypc1p reduces chronological life span. A novel finding is that, when working backwards as a ceramide synthase in vivo, Ypc1p prefers C24 and C26 fatty acids as substrates, whereas it prefers C16:0, when solubilized in detergent and working in vitro. Therefore, its physiological activity may not only concern the minor ceramides containing C14 and C16. Intriguingly, so far the sole discernable benefit of conserving YPC1 for yeast resides with its ability to convey relative resistance toward H2O2.

A novel pathway of ceramide metabolism in Saccharomyces cerevisiae.

The hydrolysis of ceramides in yeast is catalysed by the alkaline ceramidases Ypc1p and Ydc1p, two highly homologous membrane proteins localized to the ER (endoplasmic reticulum). As observed with many enzymes, Ypc1p can also catalyse the reverse reaction, i.e. condense a non-esterified fatty acid with PHS (phytosphingosine) or DHS (dihydrosphingosine) and thus synthesize ceramides. When incubating microsomes with [(3)H]palmitate and PHS, we not only obtained the ceramide PHS-[(3)H]C(16:0), but also a more hydrophobic compound, which was transformed into PHS-[(3)H]C(16:0) upon mild base treatment. The biosynthesis of a lipid with similar characteristics could also be observed in living cells labelled with [(14)C]serine. Its biosynthesis was dependent on the diacylglycerol acyltransfereases Lro1p and Dga1p, suggesting that it consists of an acylceramide. The synthesis of acylceramide could also be monitored using fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazole)-ceramides as an acceptor substrate for microsomal assays. The Lro1p-dependent transfer of oleic acid on to NBD-ceramide was confirmed by high-resolution Fourier transform and tandem MS. Immunopurified Lro1p was equally able to acylate NBD-ceramide. Lro1p acylates NBD-ceramide by attaching a fatty acid to the hydroxy group on the first carbon atom of the long-chain base. Acylceramides are mobilized when cells are diluted into fresh medium in the presence of cerulenin, an inhibitor of fatty acid biosynthesis.