Amino acid homeostasis and chronological longevity in Saccharomyces cerevisiae.

Understanding how non-dividing cells remain viable over long periods of time, which may be decades in humans, is of central importance in understanding mechanisms of aging and longevity. The long-term viability of non-dividing cells, known as chronological longevity, relies on cellular processes that degrade old components and replace them with new ones. Key among these processes is amino acid homeostasis. Amino acid homeostasis requires three principal functions: amino acid uptake, de novo synthesis, and recycling. Autophagy plays a key role in recycling amino acids and other metabolic building blocks, while at the same time removing damaged cellular components such as mitochondria and other organelles. Regulation of amino acid homeostasis and autophagy is accomplished by a complex web of pathways that interact because of the functional overlap at the level of recycling. It is becoming increasingly clear that amino acid homeostasis and autophagy play important roles in chronological longevity in yeast and higher organisms. Our goal in this chapter is to focus on mechanisms and pathways that link amino acid homeostasis, autophagy, and chronological longevity in yeast, and explore their relevance to aging and longevity in higher eukaryotes.

Whole-genome sequencing and phenotypic analysis of Bacillus subtilis mutants following evolution under conditions of relaxed selection for sporulation.

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of Bacillus subtilis evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol. 77:4105-4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of sigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by B. subtilis subsp. spizizenii and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of sigW. The results suggest that propagation of B. subtilis for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.

Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast.

We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions.

Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiae.

Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast.