Acetylation of yeast AMPK controls intrinsic aging independently of caloric restriction

Abstract

Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

Figure 1. Sip2 Is Acetylated at Four Lysine Sites by Esa1, While Rpd3 Is the Counteracting Deacetylase

(A) Cartoon of Sip2 structural domains (adapted from Hedbacker, et al. (Hedbacker and Carlson, 2008)). Mass spectrometry identified four acetylated lysine residues of Sip2, K12, K16, K17 and K256. Numbers indicate amino acid residues. Vertical straight line, myristoylation site; Downward arrows, acetylation sites; right-left arrow, regions mapped by deletion analysis as sufficient for Snf1 interaction (Amodeo et al., 2007). Ac, acetylation; N-Myr, N-myristoylation; GBD, glycogen-binding domain. (B) Chromosomally integrated sip2-3KR and sip2-4KR, but not sip2-G2A mutants, are hypoacetylated in vivo. The sip2-G2A mutant blocks myristoylation. sip2-3KR, sip2-K12/16/17R; sip2-4KR, sip2-K12/16/17/256R. (C) Rpd3, but not Hda1, removes Sip2 acetylation in vitro. Deacetylation reaction is inhibited by addition of trichostatin A (TSA). Asterisk indicates Hda1-TAP; arrowhead indicates Rpd3-TAP. (D) Sip2 is hypoacetylated in strains carrying the Ts allele of ESA1, esa1-531, but is hyperacetylated in rpd3Δ; deletion of RPD3 rescues the acetylation defect of esa1-531. (E) Increased Sip2 acetylation in rpd3Δ is blocked when lysines are mutated to arginines.

Figure 2. Sip2 Acetylation Increases the Cellular Replicative Lifespan

(A) Survival curves of the indicated strains with each median lifespan in parentheses. The fractions of live cells are plotted as a function of age in generations. Strains carrying esa1-531 exhibit lifespan shortening that are rescued by SIP2 acetylation mimetics (sip2-3KQ and sip2-4KQ). (B) Deletion of RPD3 reverses the short lifespan of esa1-531. (C) sip2-4KR mutants, as well as the sip2-G2A mutants and deletion of SIP2, decrease cellular lifespan, whereas sip2-4KQ mutants increase lifespan. (D) Simultaneous sip2-4KQ mutants on the background of rpd3Δ fail to further increase lifespan of rpd3Δ. Statistical significance was determined by Mantel-Cox log-rank test and the details are presented in Table S1.

Figure 3. Glucose Limitation and Replicative Lifespan in SIP2 Acetylation Mutants

Survival curves of WT (A), sip2Δ (B), sip2-4KR (C) and sip2-4KQ (D) grown in normal (2%, NG) vs. low glucose (0.05%, LG) media. The fractions of live cells are plotted as a function of age in generations. Median lifespan is shown in parentheses. Glucose limitation significantly increases lifespan in WT, sip2Δ, and sip2-4KR, but not in sip2-4KQ, as determined by Mantel-Cox log-rank test. The statistical results were summarized in Table S1.

Figure 4. Sip2 Acetylation and Physical Interaction between Sip2 and Snf1 Decrease as Cells Age

(A) Sip2 acetylation is significantly decreased in old cells. (B and C) Physical interaction between Sip2 and Snf1 assessed by coimmunoprecipitation is decreased in old cells (B) and esa1-531 strain but increased in rpd3Δ strain (C). (D) Interaction between Sip2 and Snf1 is decreased in sip2Δ, and deacetylation mimetics (sip2-3KR and sip2-4KR), but is not changed in sip2-G2A mutants. The interaction significantly increases in acetylation mimetics (sip2-3KQ and sip2-4KQ mutants). (E) Cellular trehalose is significantly increased when cells age. Error bars show standard error of the mean (n=3). (F) Trehalose levels are significantly higher in sip2-4KR, sip2-G2A and sip2Δ, when compared to WT; but lower in sip2-4KQ. Error bars show standard error of the mean (n=3). (G) Fraction of survival of WT, sip2-4KR, sip2-4KQ and sip2Δ, before and 1h after treatment with 1.5 mM H₂O₂, are plotted on a log scale. Error bars show standard error of the mean (n=2). Statistical significance was assessed by two-way ANOVA with post-hoc test. ns, non-significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5. Acetylation of Sip2 Affects Snf1 Kinase Activity and Results in Hypo-Phosphorylation of Sch9

(A) Snf1 phosphorylates GST-Sch9 in vitro at both serine and threonine sites. In vitro kinase assays were performed by incubating purified GST-Sch9 with or without Snf1 or ATP as indicated, and analyzed by phosphoserine antibody (α-P-Ser) and phosphothreonine antibody (α-P-Thr) to detect Sch9p phosphorylation. (B) Endogenous Sch9-HA phosphorylation decreases in snf1Δ mutants after rapamycin (200 ng/ml) treatment to suppress the TOR pathway. Arrowheads indicate the Sch9-HA band. (C) GST-Sch9 phosphorylation increases in sip2-4KR and sip2Δ, but decreases in sip2-4KQ, compared to WT after rapamycin (200 ng/ml) treatment. (D) Endogenous Sch9-HA phosphorylation significantly increases in the old cells. Arrowheads indicate the Sch9-HA band. (E) SCH9 is epistatic and thus downstream to SIP2 in regulating cellular growth. Ten-fold dilutions of the indicated strains were spotted and grown on YPD plates without (2 days, 30°C) or with rapamycin (25 ng/ml, 4 days, 30°C). (F) Deletion of SCH9 rescues the lifespan shortening of sip2Δ and sip2-4KR.

Figure 6. Sch9 Is a Parallel Downstream Target of TORC1 and Sip2-Snf1

(A–C) Comparison of growth in YPD with and without rapamycin to reveal genetic interactions between RPD3 and SIP2. Ten-fold dilutions of the indicated strains were spotted and grown on YPD plates without (2 days, 30°C) or with rapamycin (25 ng/ml, 4 days, 30°C). (D) The lifespan of sip2-4KR and sip2Δ in rapamycin is shorter than that of WT in rapamycin (25 ng/ml).

Figure 7. Model for the Effects of Sip2 Acetylation on Lifespan Regulation

Red lines indicate the intrinsic aging defense pathway identified in this study. Blue lines indicate the extrinsic nutrient-sensing pathway regulating Sch9 activity. Not shown here for clarity is evidence of a connection between the two pathways in that we also observed a modest decrease in Sip2 acetylation in glucose limitation. Ac, lysine acetylation; DeAc, lysine deacetylation; Ph, phosphorylation of serine and threonine.