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Abstract
Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.
Highlights
The prevailing and most prominent theories on the process of aging were formulated many years ago, yet the molecular basis and proximal cause(s) of aging remain largely unknown [1]
We further show that distinct factors are involved in the activation of heat shock factor 1 (Hsf1) by the different stresses; while Yap1 is required for the response to oxidative stress, Sir2 is essential for the heat shock response, and overexpression of Sir2 mimics heat shock
To gain broad insights into the effect of stationary-phase transition on Hsf1 function, we elicited three different stimuli, heat shock, oxidative stress and glucose starvation, and followed Hsf1 activity using three different reporters: one exogenously introduced, with a synthetic heat shock element (HSE) (HSE2-LacZ), and two endogenously expressed targets with native HSEs (Hsp26-GFP and Btn2-GFP). These reporters were selected because HSE2-LacZ contains 4 inverted repeats of the nGAAn/nTTCn module and is commonly used, 4 and 3 repeats are located in the BTN2 and HSP26 promoters, respectively, and both HSEs are of the perfect type [15]
Summary
The prevailing and most prominent theories on the process of aging were formulated many years ago, yet the molecular basis and proximal cause(s) of aging remain largely unknown [1] This gap in knowledge reflects the complexity of longevity, which even in unicellular model organisms such as yeast, involves many and likely interconnected intracellular pathways [2]. The replicative lifespan (RLS) is the number of daughter cells a single mother cell can produce This parameter represents the length of time a single cell remains mitotically active. The chronological lifespan (CLS) is the length of time in which stationary-phase cells remain viable, monitoring the long-term survival of non-dividing, non-mitotic cells [4]. Chronological aging can be monitored in non-dividing stationary-phase yeast [6]
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