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Life-span extension by a metacaspase in the yeast Saccharomyces cerevisiae

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Science  20 Jun 2014:
Vol. 344, Issue 6190, pp. 1389-1392
DOI: 10.1126/science.1252634

Yeast metacaspase: Grim Reaper or savior?

Yeast metacaspases are structural and possibly functional homologs of caspases that execute apoptosis—programmed cell death—in higher organisms. Malmgren Hill et al. tested whether yeast metacaspase Mca1 acts as an executioner or beneficial protein during replicative aging of yeast (see the Perspective by Kampinga). Boosting metacaspase levels caused a substantial and robust extension of life span. This lifespan extension was only partly dependent on the caspase activity of Mca1 but required the presence of the protein disaggregase Hsp104. Consistent with a role in proteostasis, Mca1 was recruited to chaperone-enriched aggregates during aging. Mca1 increased aggregate asymmetry during yeast cytokinesis and counteracted the age-associated accumulation of inclusions.

Science, this issue p. 1389; see also p. 1341

Abstract

Single-cell species harbor ancestral structural homologs of caspase proteases, although the evolutionary benefit of such apoptosis-related proteins in unicellular organisms is unclear. Here, we found that the yeast metacaspase Mca1 is recruited to the insoluble protein deposit (IPOD) and juxtanuclear quality-control compartment (JUNQ) during aging and proteostatic stress. Elevating MCA1 expression counteracted accumulation of unfolded proteins and aggregates and extended life span in a heat shock protein Hsp104 disaggregase– and proteasome-dependent manner. Consistent with a role in protein quality control, genetic interaction analysis revealed that MCA1 buffers against deficiencies in the Hsp40 chaperone YDJ1 in a caspase cysteine–dependent manner. Life-span extension and aggregate management by Mca1 was only partly dependent on its conserved catalytic cysteine, which suggests that Mca1 harbors both caspase-dependent and independent functions related to life-span control.

The cysteine-dependent aspartate-directed proteases, or caspases, are a family of proteases required for apoptosis [programmed cell death (PCD)] (13). PCD is vital for proper development, tumor suppression, immunity, and neuron homeostasis (4). The yeast Saccharomyces cerevisiae expresses a single, type I metacaspase [ancestral structural caspase homolog (5)] called Mca1 (Yca1) (6). After particular stresses, S. cerevisiae display numerous markers of PCD, dependent on the presence of Mca1 (68). Although these results imply that Mca1 is a PCD executioner protein, other reports suggest that Mca1 has beneficial functions independent of PCD, for example, in protein quality control (PQC) (913). PQC ensures that individual proteins are accurately produced, folded, compartmentalized, degraded, and prevented from aggregating (14). When, for example, during severe stress and aging, PQC fails to fully prevent protein aggregation, a second line of defense, spatial PQC, ensures that aggregates are not equally inherited during cytokinesis, a phenomenon that resets age in one cell lineage (1519). In the budding yeast S. cerevisiae, this mother cell–biased segregation of aggregated proteins requires the heat shock protein Hsp104 (20, 21).

By crossing HSP104 fused to green fluorescent protein, HSP104-GFP (22, 23), into a synthetic genetic miniarray of yeast mutants lacking chaperones or cochaperones and Hsp104 interactors (16, 22, 24), we showed that Mca1 is required for keeping yeast daughter cells free of aggregated proteins generated during a transient heat shock (fig. S1A). Constitutively elevating Mca1 levels (fig. S1B) by exchanging the weak MCA1 promoter with the strong promoter (PGPD) of the GPD gene boosted aggregate asymmetry during cytokinesis (fig. S1A). Asymmetric aggregate distribution can be achieved by limiting inheritance (retention in mother cells) or by aggregate removal (disaggregation and/or retrograde transport) in daughter cells (16, 24). These two processes were experimentally distinguished using fluorescent concanavalin A (Con A) staining of the cell wall (Fig. 1, A and B), which revealed that most genes required for establishing asymmetry (fig. S1A) were important for both aggregate removal and asymmetrical inheritance (Fig. 1C). However, deletions of MCA1 and SSA3 only affected aggregate removal (Fig. 1C). Consistently, Mca1 overproduction enhanced aggregate removal while leaving the process of inheritance unaffected (Fig. 1D).

Fig. 1 Mca1 dosage affects the establishment of aggregate asymmetry during cytokinesis.

The aggregate reporter Hsp104-GFP was used to test if the genes identified as being required for establishing aggregate asymmetry (fig. S1A) during cytokinesis were doing so by preventing aggregate inheritance or by boosting aggregate removal in daughter cells. This analysis established that Mca1 is limiting for aggregate removal in yeast daughter cells. (A) Schematic overview of the Con A staining protocol to discriminate between aggregate inheritance and/or retention (unstained buds, produced after heat shock) and removal (stained buds, present during heat shock). (B) Experimental examples depicting aggregate removal (left) and retention (right). Top rows show successful events (+), bottom rows exemplify failed processes (–), generating daughter cells containing aggregate(s). Scale bar, 5 μm. (C) Defects in aggregate removal and/or retention in asymmetry mutants identified in fig. S1A. Data are plotted as the mutants’ deviation from the wild type (WT) in aggregate inheritance (white bars; 100% increase means a twofold increase in mutant daughter cell inheritance compared with WT daughters) and removal (black bars; –100% means a twofold decrease in mutant daughter cell removal of aggregates compared with WT daughters). Data are means ± SD. N = 3 for ssa1Δ, ssa2Δ, ssa3Δ, ssb1Δ, hsp82Δ; N = 4 for ydj1Δ; N = 5 for mca1Δ. (D) Aggregate removal and inheritance in the Mca1-overproducing strain (PGPD-MCA1). Data are means ± SD. N = 3. *P < 0.05; **P < 0.005; ***P < 0.0005.

Using genome-wide synthetic genetic array (SGA) analysis (23, 25), we next identified genes that interacted negatively with MCA1, which demonstrated that Mca1 buffers against deficiencies in a few discrete functions (Fig. 2A and fig. S2A), including PQC, through negative interactions with CDC48 and RPT1 of the proteasome system. Among the nonessential genes, only one significant interaction was found: a strong negative interaction with YDJ1, which encodes the major cytosolic Hsp40 chaperone (Fig. 2, A to C). In addition, removing MCA1 in the ydj1Δ mutant exacerbated cell shape abnormalities (large, elongated buds) (Fig. 2D) and aggregate morphology (multiple amorphous aggregate structures rather than inclusion bodies) (Fig. 2E and fig. S2, B and C). Like metazoan caspases, Mca1 contains a conserved active-site cysteine [cysteine C276, previously designated C297 (6, 26)], and exchanging this single cysteine to alanine was sufficient to cause a negative genetic interaction with ydj1Δ (Fig. 2F).

Fig. 2 MCA1 displays negative genetic interactions with YDJ1 and genes involved in PQC.

To identify a possible function of Mca1 in PQC, the genetic interaction network of MCA1 was determined for both essential and nonessential genes, which demonstrated that Mca1 is buffering against deficiencies in the major Hsp40 chaperone, Ydj1, and components of the proteasome. (A) Negative genetic interactions of MCA1, clustered in functional groups, connected by known physical interactions (purple lines). (B) Growth of spores after reconstructing ydj1Δ mca1Δ (ΔΔ, red circle) double mutants by genetic crossing. (C) Growth defect of a reconstructed ydj1Δ mca1Δ (ΔΔ) double mutant. (D) Cell morphologies of the WT, ydj1Δ, and ydj1Δ mca1Δ (ΔΔ) strains. (E) Aggregate morphology after heat treatment in WT, ydj1Δ, mca1Δ, and ydj1Δ mca1Δ (ΔΔ), visualized by Hsp104-GFP. Scale bar, 5 μm. (F) Growth defect of a ydj1Δ MCA1C276A double mutant. N > 2 for all tests in (B) to (F).

In line with the suggested association of Mca1 with aggregates (11), Mca1 relocalized to Hsp104-associated cytosolic puncta during both heat stress (fig. S3A) and aging (Fig. 3A). We used the temperature-sensitive variant of the SUMO-conjugating enzyme, Ubc9ts-RFP, with red fluorescent protein (RFP), as a marker for juxtanuclear quality-control compartment (JUNQ) and the Hsp104-enriched insoluble protein deposit (IPOD) inclusions (17, 27). We found that Mca1 associated with aggregates within both these compartments (Fig. 3B) and that the JUNQ association (fig. S3B) was often close to the nucleolus (Fig. 3C) (28). During stress and aging, Mca1 relocated to inclusions independently of Ydj1, Hsp104, and Ssa1 and 2 (Fig. 3D and fig. S3C). Further, cells without Mca1, like cells lacking Ydj1, displayed increased numbers of inclusion in aged cells (Fig. 3, E and F), and age-related accumulation of inclusions was counteracted in both wild-type and ydj1Δ cells by overproducing Mca1, albeit to a higher degree in wild-type cells (Fig. 3F). The IPOD-specific reporter Rnq1 (27) revealed that most aggregates in old cells were IPODs and that Mca1 overproduction caused a 30% reduction in the accumulation of these inclusions (fig. S3, D and E). To test by which route Mca1 may prevent the buildup of such protein inclusions, we determined the effect of Mca1 dosage on luciferase folding (29) and the levels of the signal sequence–deficient, unfolded carboxypeptidase Y, CPY*, fused to Leu2 (hereafter called ΔssCL*) (30, 31). We found that neither Mca1 overproduction nor an mca1Δ deletion affected folding activity (fig. S3F). In contrast, Mca1 overproduction reduced, whereas mca1Δ increased, the levels of the unfolded proteasome substrate ΔssCL* (Fig. 3, G and H), which suggested that Mca1 is required for efficient removal of terminally unfolded proteins.

Fig. 3 Mca1 localizes to IPOD and JUNQ quality-control compartments and prevents accumulation of unfolded proteins and aggregates formed upon aging.

(A) Colocalization of Mca1 and Hsp104 at aggregates in replicatively aged cells (12 generations). The fraction (%) of cells displaying colocalization is indicated near the micrograph; 400 cells were analyzed. N > 2. (B) Colocalization of Mca1 and Ubc9ts at peripheral (IPOD) and juxtanuclear (JUNQ) compartments [4′,6′-diamidino-2-phenylindole (DAPI)–stained nucleus in blue]. The fraction (%) of Mca1 foci colocalizing with Ubc9ts is indicated near the micrograph; 188 cells were analyzed. N > 2. (C) Mca1 localization to aggregates at peripheral sites and the nucleolus, visualized by Sik1-RFP. The fraction (%) of Mca1 foci colocalizing with Sik1 is indicated near the micrograph; 245 cells were analyzed. N > 2. (D) Mca1 association with aggregates in the absence of SSA1, SSA2, HSP104, and YDJ1 as indicated. N > 2. (E) Visualization of Hsp104-GFP aggregates in young and replicatively aged cells as indicated with (+) and without (–) overexpression of MCA1 (MCA1 OE). Scale bars, 5 μm. (F) Percentage of cells from (E) containing aggregates in young (0 generations) and aged cells (13 generations for WT and mca1Δ, 9 generations in ydj1Δ strains). N = 6 for mca1Δ and WT MCA1 OE, N = 3 for ydj1Δ and ydj1Δ MCA1 OE. Data are means ± SD. *P < 0.05; ***P < 0.0005. (G) The ΔssCL* protein that misfolds in the cytoplasm and is degraded by the proteasome (30, 31) was used to test if Mca1 is involved in facilitating removal of misfolded proteins. Cells expressing ΔssCL* (ΔssCPY* fused to Leu2) were spotted on indicated media and incubated for 3 days at 30°C. Enhanced growth on plates lacking leucine indicates stabilization of ΔssCL* (30, 31), whereas reduced growth indicates increased degradation. N = 3. (H) Immunoblotting showing relative ΔssCL* levels of the indicated strains. Data are means ± SD. N = 3.

In view of these data, we tested whether MCA1 was acting as a pro- (executioner) or anti- (gerontogene) aging gene. The lack of Mca1 had little effect on the replicative life span in otherwise wild-type cells but accelerated aging of cells lacking Ydj1 (Fig. 4A), which confirmed that Mca1 becomes indispensable in the absence of Ydj1. Overproducing Mca1 extended life span (46 to 56%) in a manner partly dependent on Ydj1 (Fig. 4, B and C). That life-span extension by Mca1 overproduction was less efficient in cells lacking Ydj1 is consistent with a more pronounced reduction of aggregates in the former (Fig. 3F) and suggests that proper protein homeostasis is required to achieve full effects of elevated Mca1 levels. Indeed, overproduction of Mca1 did not result in a statistically significant extension of life span in the absence of Hsp104 (Fig. 4D) or a reduction in proteasome levels by deleting the proteasome regulator Rpn4 (32) (Fig. 4E). Thus, the removal of unfolded and aggregated proteins is a key feature of Mca1’s role in life-span control.

Fig. 4 Mca1 extends life span and prevents aggregate accumulation in a partially caspase cysteine–independent manner.

To test if Mca1 acts as an executioner gene or gerontogene during the life history of yeast mother cells, the replicative life span of Mca1-deficient and Mca1-overproducing cells was determined. This analysis demonstrated that Mca1 acted as a life-span–extending gene and that life-span extension required both Hsp104-dependent disaggregase activity and fully functional proteasomes linking Mca1-dependent life-span control to the removal of damaged and aggregated proteins. (A) Replicative life span of WT, ydj1Δ, mca1Δ, and ydj1Δ mca1Δ. Life span of ydj1Δ is shorter than that of WT (P = 5.0E-4; 50 cells) and is further shortened when also deleting MCA1 (P = 5.1E-5; 50 cells). No difference was seen between mca1Δ and WT (P = 0.99; 80 cells). (B and C) Life-span extension by Mca1 overproduction in cells lacking YDJ1 (B) (P = 3.00E-2; 80 cells), and in WT cells (C) (P = 0.037; 80 cells). (D and E) Life span of the Mca1-overproducing strain lacking the disaggregase Hsp104 (P = 0.014; 80 cells) (D) and the proteasome regulator Rpn4 (E) (P = 0.16; 64 cells). (F) Mca1 proteolytic processing during aging and H2O2 exposure in cells overproducing Mca1 or the caspase-inactive Mca1C276A. Red arrow indicates the 12-kD product typically cleaved off upon caspase activation. (G) Age-induced aggregation in cells overproducing caspase-inactive Mca1C276A. Data are means ± SD. N = 3. **P < 0.005. (H and I) Effect of overproducing the caspase-inactive Mca1C276A on the life span of WT (H) (P = 3.5E-5; 64 cells) and ydj1Δ (I) (P = 0.0082; 64 cells) cells.

The conserved catalytic cysteine-histidine dyad of Mca1 is, like metazoan caspases, activated under certain conditions including H2O2 exposure, to autocatalytically remove a small ∼12-kD subunit on the Mca1 protein (6, 33). We found that this 12-kD subunit accumulated in aged cells carrying the PGPD-MCA1 fusion, similar to cells exposed to H2O2 (Fig. 4F), which indicated that typical metacaspase processing is triggered upon mother cell aging. The Mca1C276A protein was, when overproduced, incapable of removing the 12-kD subunit during aging (Fig. 4F) and partially defective in counteracting aggregate accumulation (Fig. 4G) and extending life span (Fig. 4H). Overproduction of Mca1C276A in cells lacking Ydj1 reduced rather than extended life span (Fig. 4I), consistent with the requirement for the active-site cysteine in Ydj1-deficient cells (Fig. 2F). Thus, both cysteine-dependent and independent functions of Mca1 are required for life-span control of which the cysteine-independent one can only be realized in cells harboring functional Ydj1.

Here, we have shown that Mca1 has a beneficial cell-autonomous function in PQC that can compensate for the lack of the major Hsp40 chaperone of the yeast cytosol and facilitate the removal of an unfolded protein, counteract accumulation of protein aggregates, and prolong cellular life span in an Hsp104 disaggregase- and proteasome-dependent manner. The fact that the Hsp104 disaggregase is required for Mca1 to extend life span suggests that protein aggregates and/or inclusions are true aging factors in the yeast model system. Because the beneficial functions of Mca1 related to aggregate management and longevity are only partially dependent on the catalytic cysteine C276, it is conceivable that under some conditions, metacaspase-dependent PCD activities (68) may be balanced by both metacaspase-dependent and independent proteostasis. In addition, the data raise the possibility that caspases and/or metacaspases originally evolved as PQC-related cytoprotective factors that were later adopted as PCD-related executioners, perhaps upon their overinduction during severe stress. Elucidating the nature of the environmental cues regulating the switching of metacaspase functions between proteostasis and PCD might explain how decisions concerning survival are made at the level of the individual cell versus the cell community.

Supplementary Materials

www.sciencemag.org/content/344/6190/1389/suppl/DC1

Materials and Methods

Figs. S1 to S3

Table S1

References (3439)

References and Notes

  1. Materials and methods, figs. S1 to S3, and table S1 are available as supplementary materials on Science Online.
  2. Acknowledgments: The authors thank C. Boone for providing strain collections for the SGA analysis, L. Megeney for the MCA1C276A strain and the pLM1010 plasmid, W.-K. Huh for the pSIK1-RFP plasmid, D. Kaganovich for the pUBC9ts-RFP plasmid, D. Cyr for the pRS423-Cup1-Rnq1-mRFP plasmid, E. Deuerling for the Mca1 antibody, C. Andréasson and U. Hartl for the luciferase plasmids, F. Eisele for the pFE15 (CPY) plasmid, and A. Sigurdsson for assistance in complementation assays. This work was supported by grants from the Swedish Natural Research Council (VR) (T.N. and B.L.) and the Knut and Alice Wallenberg Foundation (Wallenberg Scholar) and European Research Council (Advanced Grant; QualiAge) to T.N., the Swedish Cancer Society (CAN 2012/601) and Stiftelsen Olle Engkvist Byggmästare Foundation to B.L.
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