Induction of Sis1 promotes fitness but not feedback in the heat shock response

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR. However, recent work has implicated newly synthesized proteins (NSPs) – rather than unfolded mature proteins – and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs s...,
Strain construction                        

Yeast strains and plasmids used in this study are listed in Table S1. All strains are derived from the W303 parent strain. CRISPR-mediated promoter swapping was performed to create the 1xSup35pr-Sis1 (1701) and 1xSup35pr-Sis1-P2A-mscarlet (1661) strains. CRISPR-Cas9 mediated precise, scarless replacement of the native Sis1 promoter with the 600-bp Sup35 promoter. To construct the final non-inducible Sis1 line (2xSup35pr-Sis1, 1761), we incorporated a second copy of Sup35pr-Sis1 at the tryp locus.
In all other cloned lines, genes were tagged at the endogenous locus. Cells were transformed with double-stranded DNA fragments containing ~20 bp homologous flanking regions. This method takes advantage of homology-directed repair mechanisms in S.cerevisiae, as described previously (Longtine et al., 1998).
Cell Growth
For heat shock time courses followed by flow cytometry, cells were cultured in 1xSDC media overnight at room temperature (synthetic ...,