The 'programmable' features of the N-end rule degradation pathway and a ubiquitin fusion strategy were exploited to create a family of destabilized cyan fluorescent proteins (CFP) to be used as transcriptional reporters. The N-degron CFP reporters characterized in this report have half-lives of approximately 75, 50 and 5 min, but further modification of the N-degron signal sequences could readily generate additional variants within this range. These destabilized CFP reporters have been engineered into convenient plasmid constructs with features to enable their expression from upstream activating sequences of choice and to facilitate their targeted integration to the URA3-TIM9 intergenic region of chromosome V. The advantages and limitations of these reporters as temporal indicators of gene expression in living cells are illustrated by their application as reporters of galactose- and pheromone-induced transcription. The plasmid design we describe and the range of different stabilities that are theoretically feasible with this strategy make the N-degron CFP reporters easily adapted to a variety of applications.

Mitogen-activated protein kinase kinase kinase-Ste11 (MAPKKK-Ste11), MAPKK-Ste7, and MAPK-Kss1 mediate pheromone-induced mating differentiation and nutrient-responsive invasive growth in Saccharomyces cerevisiae. The mating pathway also requires the scaffold-Ste5 and the additional MAPK-Fus3. One contribution to specificity in this system is thought to come from stimulus-dependent recruitment of the MAPK cascade to upstream activators that are unique to one or the other pathway. To test this premise, we asked if stimulus-independent signaling by constitutive Ste7 would lead to a loss of biological specificity. Instead, we found that constitutive Ste7 promotes invasion without supporting mating responses. This specificity occurs because constitutive Ste7 activates Kss1, but not Fus3, in vivo and promotes filamentation gene expression while suppressing mating gene expression. Differences in the ability of constitutive Ste7 variants to bind the MAPKs and Ste5 account for the selective activation of Kss1. These findings support the model that Fus3 activation in vivo requires binding to both Ste7 and the scaffold-Ste5 but that Kss1 activation is independent of Ste5. This scaffold-independent activation of Kss1 by constitutive Ste7 and the existence of mechanisms for pathway-specific promoter discrimination impose a unique developmental fate independently of any distinguishing external stimuli.