Amino acid homeostasis and chronological longevity in Saccharomyces cerevisiae

Abstract

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.

Fig. 8.1

General amino acid control is not required for extension of CLS by CR or rapamycin. CLS measurements were done with yeast strains in the BY4742 genetic background (MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) grown in synthetic dextrose (SD) minimal medium as described (Alvers et al. 2009). Viability is expressed in terms of colony forming units (CFU) per mL and is plotted as the log of the percent viability on day three of the experiment. Wild type (WT, hoΔ) and gcn4Δ strains were grown in: normal (2%) glucose SD; low (0.4%) glucose SD; normal glucose SD followed by washing with water beginning on day three (Water); or normal glucose SD plus 10 nM rapamycin (+Rap)

Fig. 8.2

Macroautophagy is up-regulated in a leu3Δ strain. Activation of autophagy in leu3Δ and wild type (WT) strains was measured over 5 days of a CLS experiment performed as described (Alvers et al. 2009). Strains in the BY4742 background were transformed with plasmid pCuGF-PAUT7(416) (URA3), grown in synthetic dextrose medium (Sherman 2002) with standard (SD) or three-fold elevated levels (SD+HKL) of histidine, lysine, and leucine, and a western blotting assay was used to measure cellular levels of GFP and the GFP-Atg8p fusion protein (Klionsky et al. 2007). The percent conversion of GFP-Atg8p to GFP on each day reflects the extent of autophagic activation and is plotted in graphs below western blots. Equivalent amounts of cells, based on cell density measurements, were collected, processed, and analyzed on blots. Day zero samples were collected during mid-log growth. Quantification was done using ImageJ software [% processing = 100 × GFP ÷ (GFP + GFP−Atg8p)]

Fig. 8.3

Role for nitrogen assimilation in chronological longevity. a Nitrogen / ammonia assimilation pathway in S. cerevisiae. The TCA cycle intermediates are oxaloacetate (O), citrate (C), cis-aconitate (A), isocitrate (I), 2-ketoglutarate (2-kG), and succinate (S). Gene products are discussed in the text. AA, amino acids. P, purines and pyrimidines. Consumption of ATP, NAD⁺, and NADP⁺ are omitted for simplicity. Inhibition by TORC1 and Ure2p are diagrammed separately but participate in nitrogen catabolite repression (NCR). Interactions between NCR and the retrograde signaling pathway as well as negative regulation by Dal80p and Deh1p are not shown. b and c CLS measurements were done using deletion mutants in the BY4742 genetic background grown in SD minimal medium as described in Fig. 8.1. Viability is expressed in terms of colony forming units (CFU) per mL of culture and is plotted as the log of the percent viability on day three of the experiment. The results shown are representative of at least two independent experiments