Cells respond to a plethora of signals using a limited set of intracellular signal transduction components. Surprisingly, pathways that transduce distinct signals can share protein components, yet avoid erroneous cross-talk. A highly tractable model system in which to study this paradox is the yeast Saccharomyces cerevisiae, which harbors three mitogen-activated protein kinase (MAPK) signal transduction cascades that share multiple signaling components. In this review we first describe potential mechanisms by which specificity could be achieved by signaling pathways that share components. Second, we summarize key features and components of the yeast MAPK pathways that control the mating pheromone response, filamentous growth, and the response to high osmolarity. Finally, we review biochemical analyses in yeast of mutations that cause cross-talk between these three MAPK pathways and their implications for the mechanistic bases for signaling specificity. Although much remains to be learned, current data indicate that scaffolding and cross pathway inhibition play key roles in the maintenance of fidelity.
The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway. Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273. Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro. Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases. Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth. Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity.
Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.
Summary Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. We have identified a single amino acid substitution in the Caulobacter Structural Maintenance of Chromosomes (SMC) protein that disrupts chromosome segregation and cell division. The E1076Q point mutation in the SMC ATPase domain caused a dominant-negative phenotype in which DNA replication was able to proceed, but duplicated parS centromeres, normally found at opposite cell poles, remained at one pole. The cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere, but chromosomes remained unsegregated and appeared to be stacked upon one another. Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. We propose that SMC spuriously links the duplicated chromosome immediately after passage of the replication fork. In wild type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. The loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both chromosomes, inhibiting segregation.
The yeast kinase scaffold Ste5 has been proposed to prevent unwanted cross-talk between the pheromone response pathway and other MAPK cascades. Protein fusion experiments have demonstrated that covalently tethering signaling components to each other or to Ste5 can determine the outcome of signaling. However, these do not fully test the role of scaffolds in signaling specificity, since fusing components precludes differential dissociation of subpopulations. We performed a targeted genetic screen on STE5 and repeatedly identified recessive mutations in a conserved residue, E756, in the Ste7/MEK-binding domain that caused erroneous activation of the filamentation MAPK pathway by pheromone signaling. Mutant cells exhibited a shift in the MAPK activation pattern such that the filamentation MAPK Kss1 was predominately activated in response to pheromone. Velocity sedimentation studies showed that the mutant scaffold was defective in binding to a phosphorylated subpopulation of Ste7. Our data suggest that increased dissociation of activated Ste7 kinase from the mutant scaffold may cause the observed shift in MAPK activation from Fus3 to Kss1 and the resulting loss of specificity. Cross-talk in ste5-E756G cells was due to both increased activation of Kss1 and reduced Fus3-dependent degradation of the filamentation pathway transcription factor Tec1. These studies demonstrate a role for an endogenous scaffold in signaling specificity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.