Here we report that a wide range of SDS-insoluble proteins accumulate in S. cerevisiae during postmitotic aging. These insoluble proteins are absent in logarithmically growing or young postmitotic cells. We find that the ensemble of proteins that become insoluble in postmitotic yeast is similar to that that accumulates during aging in adult C. elegans. This indicates that the types of proteins that undergo this conformation transition in response to age are conserved ( and Supplemental Table S1). Furthermore, this shows that the causative factors and/or mechanisms governing age-dependent accumulation of insoluble proteins are conserved between fungi and nematodes and thus may be conserved in eukaryotes in general.
Recently an independent group identified proteins that became insoluble with age in C. elegans
. Using techniques similar to those used in our yeast and nematode aging studies, David et al. (2010
) identified 461 proteins that became insoluble with age in C. elegans
. Although there is significant overlap with the proteins that we identified in aging C. elegans
with those reported in the study by David and colleagues (Reis-Rodrigues et al., 2012
), we note that there is also remarkable similarity with the age-dependent insoluble yeast proteins identified in this study. Of the 246 insoluble yeast proteins that have nematode homologues, 102 (41.5%) were found to accumulate in aged nematodes in the study carried out by David et al. (2010
). Overall the similarity of the content of the age-dependent insoluble protein fraction found in distinct aging models in divergent species strongly supports the idea that this is a regulated and conserved phenomenon that is not unique to the yeast chronologic aging paradigm.
We show that insoluble protein only accumulates in the nonquiescent subpopulation of postmitotic yeast. This suggests that insoluble protein accumulation in the postmitotic cells is not merely due to nutrient depletion in stationary phase cultures. The quiescent subpopulation has no detectable insoluble protein, yet both subpopulations were subjected to the same nutrient conditions. Instead, we suggest that insoluble protein accumulation is related to the physiological state of the nonquiescent cells (Aragon et al., 2008
). In this case, insoluble protein accumulation is another indication of impaired protein homeostasis in nonquiescent cells, yet the role of insoluble protein inclusions in this context remains unclear. Recently nonquiescent cells were shown to have lower levels of mitochondrial proteins as measured by fluorescence intensity in strains carrying single GFP fusions of mitochondrial-localized proteins (Davidson et al., 2011
). The nonquiescent fraction was also shown to have lower levels of respiration, higher levels of reactive oxygen species, and higher rates of petite formation, all indicative of mitochondrial dysfunction (Davidson et al., 2011
). Given that we find insoluble protein containing a significant amount of mitochondrial proteins in the same subpopulation of cells, we suggest that the lower levels of mitochondrial proteins in nonquiescent cells are due to their sequestration into the insoluble fraction. Thus, if insoluble protein accumulation is an active process that occurs in nonquiescent cells (discussed later), it may be a driving factor in decreasing mitochondrial function and the associated phenotypes of nonquiescent cells.
Asymmetrical inheritance of aggregated and or damaged proteins in yeast has been well documented and described as a form of “spatial protein quality control” (Nystrom, 2011
). This phenomenon has been shown to involve a number of active processes dependent upon chaperones, deacetylases, and cytoskeletal proteins (Aguilaniu et al., 2003
; Liu et al., 2010
). We observed that SDS-insoluble protein is present in nonquiescent cells and absent in quiescent cells. The nonquiescent cell population comprises the mother cells from the last cell mitotic division upon entrance into stationary phase, whereas the quiescent cells are the daughters from this division (Allen et al., 2006
). The difference in insoluble protein distribution may be a result of asymmetrical inheritance of this protein fraction during this final division and could contribute to the enhanced viability of the quiescent daughter cells in the postmitotic population.
Postmitotic aging is not the only trigger for the formation of SDS-insoluble protein. We found that either nutrient limitation or TORC1 inactivation is sufficient to induce this process ( and ). Our observations indicate that TORC1 activity suppresses insoluble protein accumulation. Supporting this idea, we find that insoluble protein accumulation correlates with activation of the canonical autophagic pathway ( and ), which is also negatively regulated by TORC1. However, insoluble protein accumulation does not require activation of autophagy (). This indicates that TORC1 modulates insoluble protein accumulation and activation of autophagy through distinct mechanisms. Given that more than half of the insoluble proteins in yeast belong to complexes that have been observed in ABs and are known substrates of autophagy (; Baba et al., 1994b
; Kraft et al., 2008
; Kanki et al., 2009
; Okamoto et al., 2009
), we suggest that insoluble protein accumulation plays a role in autophagic cargo preparation (). This idea is analogous to the sequestration of damaged proteins into aggresomes for degradation via autophagy (Taylor et al., 2003
; Wang et al., 2009
). Although aggresomes have been studied primarily in the context of misfolded proteins that accumulate in specific neurodegenerative diseases (i.e., expanded polyglutamine proteins), our work suggests that formation of SDS-insoluble proteins occurs during nutrient limitation, TORC1 inactivation, and postmitotic aging. Overall TORC1 regulation of cargo sequestration and autophagic degradation suggests that these mechanisms work in concert to promote protein degradation, amino acid recycling, and protein homeostasis.
The regulation of protein aggregation has been extensively studied in several models of aging and disease. A study in C. elegans
reported that aggregation of the Aβ peptide into an insoluble form is promoted by a daf-16–
dependent mechanism activated when proteasome capacity is overwhelmed (Cohen et al., 2006
). This report suggests that active promotion of insoluble protein aggregation is an important mechanism facilitating protein homeostasis and is regulated in concert with disaggregase activity to promote protein degradation via the ubiquitin-proteasome pathway (Cohen et al., 2006
). In studies using cell models of neurodegenerative diseases, the regulated formation of large insoluble protein aggregates has been correlated with enhanced survival (Arrasate et al., 2004
; Cohen et al., 2006
; Ben-Zvi et al., 2009
). Overall these observations underscore the importance of regulating protein homeostasis through mechanisms that modulate protein conformation and solubility. The results presented in this study indicate that Tor1 plays a central role in regulation of protein solubility during aging and in response to nutrient stress. It is known that reduction of Tor1 activity by either genetic mutation or rapamycin treatment can increase longevity in yeast, fly, and mouse models (Kapahi et al., 2004
; Kaeberlein et al., 2005
; Powers et al., 2006
; Harrison et al., 2009
). Our results suggest that Tor1-dependent regulation of protein solubility and aggregation is likely to play a significant role in protein homeostasis, longevity, and disease.