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Yeast mitogen-activated protein kinase (MAPK) signaling pathways transduce external stimuli into cellular responses very precisely. The MAPKs Slt2/Mpk1 and Hog1 regulate transcriptional responses of adaptation to cell wall and osmotic stresses, respectively. Unexpectedly, we observe that the activation of a cell wall integrity (CWI) response to the cell wall damage caused by zymolyase (␤-1,3 glucanase) requires both the HOG and SLT2 pathways. Zymolyase activates both MAPKs and Slt2 activation depends on the Sho1 branch of the HOG pathway under these conditions. Moreover, adaptation to zymolyase requires essential components of the CWI pathway, namely the redundant MAPKKs Mkk1/ Mkk2, the MAPKKK Bck1, and Pkc1, but it does not require upstream elements, including the sensors and the guanine nucleotide exchange factors of this pathway. In addition, the transcriptional activation of genes involved in adaptation to cell wall stress, like CRH1, depends on the transcriptional factor Rlm1 regulated by Slt2, but not on the transcription factors regulated by Hog1. Consistent with these findings, both MAPK pathways are essential for cell survival under these circumstances because mutant strains deficient in different components of both pathways are hypersensitive to zymolyase. Thus, a sequential activation of two MAPK pathways is required for cellular adaptation to cell wall damage. INTRODUCTIONSaccharomyces cerevisiae yeast cells are exposed to rapid and extreme changes in the environment. In response to these changes, precise responses are coordinated by the cell through different mitogen-activated protein kinase (MAPK) signaling pathways. In this sense, external cues are transduced into appropriate cellular responses, allowing cells to adapt to particular environmental conditions. In budding yeast, four MAPKs, Fus3, Kss1, Hog1, and Slt2/Mpk1, control mating, filamentation/invasion, high osmolarity, and cell integrity pathways, and they are activated in response to mating pheromones, starvation, osmolarity, and cell wall damage, respectively (Qi and Elion, 2005).Yeast cell integrity depends on a particular external envelope: the cell wall, which is necessary not only for maintaining cell morphology but also for protecting cells from extreme conditions. The components of this structure form a macromolecular complex whose mechanical strength allows cells to support turgor pressure against the plasma membrane (Levin, 2005;Lesage and Bussey, 2006). Because of the importance of the cell wall for survival, stress conditions that alter this structure lead to the activation of a cellular response that has been called the "compensatory mechanism" (Popolo et al., 2001). This response is triggered by the cell in an attempt to survive, and it is characterized by 1) an increase in ␤-glucan and chitin contents; 2) changes in the association between cell wall polymers; 3) an increase in the amount of several cell wall proteins (CWPs); and 4) the relocalization of important proteins from the cell wall construction machinery to the later...

The genome of coronaviruses, including SARS‐CoV‐2, encodes for two proteases, a papain like (PLpro) protease and the so‐called main protease (Mpro), a chymotrypsin‐like cysteine protease, also named 3CLpro or non‐structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS‐CoV, SARS‐CoV‐2, and hCoV‐NL63 coronviruses, an LC‐MS based N‐terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS‐CoV‐2 Mpro, 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS‐CoV and hCoV‐NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.