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. 2009 Aug;8(4):353-69.
doi: 10.1111/j.1474-9726.2009.00469.x. Epub 2009 Apr 21.

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

Ashley L Alvers  1 , Laura K Fishwick, Michael S Wood, Doreen Hu, Hye S Chung, William A Dunn Jr, John P Aris
Affiliations

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

Ashley L Alvers et al. Aging Cell. 2009 Aug.

Abstract

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.

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Figures

Figure 1
Figure 1
Autophagy is required for chronological longevity in synthetic media. CLS was measured by determining cell viability over time in synthetic dextrose minimal medium (SD, panel A), two different synthetic complete media (SC1 and SC2, panels B and C), and non-defined rich medium (YPD, panel D). SC1 refers to Sherman’s formulation (Sherman, 2002) and SC2 refers to Fink’s formulation (Amberg et al., 2005; Styles, 2002) (see Table 2). atg1Δ and atg7Δ strains are deficient in autophagy. The atg11Δ strain carries out autophagy, but is deficient in pexophagy and the CVT pathway. The WT strain is “wild type” for autophagy. Viability is expressed in terms of colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1. Two independent experiments are shown.
Figure 2
Figure 2
Chronologically aging yeast do not exhibit senescence prior to cell death. CLS in synthetic dextrose minimal medium was determined using two measures of cell viability: colony forming units (CFU) (panel A) and staining with the vital dye FUN-1 (panel B). Yeast strains competent (WT, atg11Δ) or deficient (atg1Δ, atg7Δ) in autophagy were analyzed. Viability is expressed in terms of the percent of viability on day 1 and is plotted on a linear scale over a 5-day time period. FUN-1 staining was done as described in Materials and Methods. Cell density (OD600) measurements are shown (panel C).
Figure 3
Figure 3
Autophagy is required for chronological longevity in the W303 strain background. CLS was measured by determining cell viability over time in synthetic dextrose minimal medium. The W303 parental strain is competent for autophagy. atg1Δ (W) and atg7Δ (W) are autophagy deficient strains constructed in the W303 parent. The atg11Δ (W) strain carries out autophagy, but is deficient in the pexophagy and the CVT pathway. Viability in colony forming units (CFU) per ml culture is plotted as the log of percent viability on day 1.
Figure 4
Figure 4
Specific non-essential amino acids promote chronological longevity. The CLS of autophagy-deficient (atg1Δ, atg7Δ) yeast was measured in synthetic dextrose minimal medium containing individual non-essential amino acids (I, T, V, D, E, F, R, S, W, M, or Y) or adenine (a) added to final concentrations as described in Table 2. Viability in colony forming units (CFU) per ml of culture is plotted as the log of the percent of viability on day 2.
Figure 5
Figure 5
Non-essential amino acids isoleucine, threonine, and valine promote chronological longevity. The CLS of yeast strains competent (WT, atg11Δ) or deficient (atg1Δ, atg7Δ) in autophagy was measured in synthetic dextrose (SD) minimal or synthetic complete (SC1) media prepared as described in Table 2. Isoleucine (I), threonine (T), and valine (V) were added individually or in combination (ITV) to SD medium to achieve final concentrations present in SC1 medium (Table 2). Viability is expressed in colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 3.
Figure 6
Figure 6
Constitutive expression of Gcn4p suppresses longevity conferred by non-essential amino acids isoleucine, threonine, and valine. Yeast strains competent (WT, atg11Δ) or deficient (atg1Δ, atg7Δ) in autophagy were transformed with three plasmids: YEp50 (vector control), p164 (GCN4), or p238 (GCN4C, which constitutively expresses Gcn4p). CLS was measured in synthetic dextrose minimal media without (SD) or with the non-essential amino acids isoleucine, threonine, and valine (SD + ITV) added to final concentrations present in SC1 (see Table 2). Viability is expressed in terms of colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1.
Figure 7
Figure 7
Non-essential amino acids isoleucine, threonine, and valine suppress GCN4 expression. The WT strain was transformed with plasmids p164 (GCN4) or p238 (GCN4C, which constitutively expresses Gcn4p) and CLS was measured in synthetic dextrose minimal medium without (SD) or with (SD + ITV) the non-essential amino acids isoleucine, threonine, and valine added to final concentrations listed for SC1 (Table 2). A. Viability is expressed in colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1. B. Cell density (OD600) was measured on days 0, 1, 3, 5, and 8. C. Measurement of Gcn4p levels was done during chronological aging. WT cells transformed with p164 (GCN4) or p238 (GCN4C) and grown in SD or SD + ITV were harvested on days 0, 1, or 3. Equal amounts of whole cell lysates (based on OD600 units) were analyzed by Western blotting with an affinity-purified antibody to Gcn4p (see Materials and Methods). The intensities of Gcn4p bands (arrowhead) were quantified using ImageJ software and normalized to the intensities of bands (asterisk) detected non-specifically in a gcn4Δ strain (Δ). Values for the normalized Gcn4p band intensities relative to lane 8 are shown below each lane (Gcn4p). Positions of molecular weight markers are shown on the left (in kDa).
Figure 8
Figure 8
Essential amino acids promote chronological longevity. The CLS of yeast competent (WT, atg11Δ) or deficient (atg1Δ, atg7Δ) in autophagy was measured in synthetic dextrose minimal medium containing histidine, lysine, leucine, and uracil supplements added to standard (SD) or three-fold elevated (3X HKLu) final concentrations as described in Table 2. Viability is expressed in terms of colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1.
Figure 9
Figure 9
Leucine promotes chronological longevity. The CLS of autophagy-competent (WT, atg11Δ) or autophagy-deficient (atg1Δ, atg7Δ) yeast strains was measured in synthetic dextrose minimal media containing standard amounts of supplements (SD), a three-fold elevated level of leucine (3X L), a three-fold elevated levels of histidine, lysine, and uracil (3X HKu), or 10 mM dibasic potassium phosphate (SD + PO4) (Table 2). Transformants carrying low-copy number, centromeric plasmids pRS513 (HIS3), pRS315 (LEU2), pRS316 (URA3), or pRS317 (LYS2) were also analyzed. Viability is expressed in terms of colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1.
Figure 10
Figure 10
LEU2 promotes chronological longevity and blocks the effects of constitutive expression of Gcn4p. LEU2 strains competent (WT, atg11Δ) or deficient (atg1Δ, atg7Δ) in autophagy were transformed with three plasmids: YEp50 (vector control), p164 (GCN4), or p238 (GCN4C, which constitutively expresses Gcn4p). The LEU2 strains YAA1, YAA3, YAA5, and YAA7 are described in Table 1. CLS was measured in synthetic dextrose minimal media without (SD) or with (SD + ITV) the non-essential amino acids isoleucine, threonine, and valine added to final concentrations present in SC1 (Table 2). Viability is expressed in terms of colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 3.
Figure 11
Figure 11
Deletion of the regulatory gene LEU3 extends CLS. Control (WT), leu3Δ, and LEU2 (YAA1) strains (Table 1) were grown in synthetic dextrose minimal medium (SD) with standard or three-fold elevated levels of leucine (3X L) (Sherman, 2002, Table 2). CLS was measured and viability is expressed in terms of colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1 or day 3.
Figure 12
Figure 12
Induction of autophagy during chronological aging. Control (WT) and LEU2 (YAA1) strains were transformed with plasmid pCuGFPAUT7(416) (Kim et al., 2001), which expresses a GFP-Atg8p fusion protein that is proteolytically processed in an autophagy-dependent manner to yield GFP. Transformants were grown in synthetic dextrose minimal medium containing standard amounts of the essential amino acids histidine, lysine, and leucine (SD) or containing three-fold increased amounts of the same supplements (SD + 3X HKL) as described in Table 2. Protein lysates from cells collected on days 0–5 were analyzed by western blotting using a GFP-specific polyclonal antibody (see Materials and Methods). Blots were reprobed with monoclonal antibody directed against the nucleolar protein Nop1p, which served as a loading control. Cell density (OD600) and percent viability values are shown below the lane corresponding to the day on which data were collected.
Figure 13
Figure 13
Amino acid homeostasis and chronological longevity in yeast. A. Model highlighting relationships between amino acid homeostasis and chronological longevity in yeast. De novo synthesis, uptake, and autophagy contribute to the cellular amino acid (AA) pools, including the branched side chain amino acids leucine (L), isoleucine (I), and valine (V). The superpathway for branched side chain amino acids is regulated by the Leu3p transcription factor, which functions to balance synthesis of L, I, and V. Yeast strains containing a leu2 mutation are hypothesized to misregulate this superpathway in a manner involving Leu3p. Branched side chain amino acids from uptake or autophagic recycling compensate for superpathway misregulation in a manner dependent on Gcn4p, which mediates general amino acid control and regulates levels of Leu3p. Not depicted are pathways by which Gcn4p regulates de novo synthesis and uptake of amino acids and autophagy. Panel B. Bar graph of codon abundance in yeast (in percent of total codons). Data were derived from a codon usage table generated by CUSP (EMBOSS software suite). The FASTA dataset for ORFs (not including dubious ORFs and pseudogenes) was obtained from the Saccharomyces Genome Database. Similar results were obtained using the FASTA dataset for all ORFs, or publicly available codon usage tables prepared by J. M. Cherry that were generated using the GCG program CodonFrequency based on ORFs listed within SGD as of January 1999 (data not shown).
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