Conservation of downstream components in the pheromone and hyper-osmolar glycerol pathways in S. cerevisiae. (a) The pheromone signal results in phosphorylation of the MAPKK Ste7, which phosphorylates the MAPK Fus3 aided by a scaffolding protein Ste5 (ref. 23). Phosphorylated Fus3 in turn activates the transcription factor Ste12 by relieving its repression by Dig1 and Dig2 (ref. 24), and activates Far1 leading to cell-cycle arrest. Activated Ste12 initiates the transcription of mating-specific genes including FUS1. The stimulation of cells with an osmolyte (for example, sorbitol) results in phosphorylation of the MAPKK Pbs2, which phosphorylates the MAPK Hog1, which in turn activates several transcription factors (including Hot1). This initiates the transcription of several genes, including STL1. (b) Matrices corresponding to each protein in a encode the pair-wise percentage identity between the amino-acid sequences of all orthologues of that protein. The matrix for Hog1 is enlarged and labelled. The outlined matrix element in the Hog1 matrix shows the percentage identity between the Hog1 orthologues in A.gossypii and S. castelli. As the matrices are symmetrical, only half of each is shown. Beside each matrix is the substitution ratio (N/S) measuring the evolution rate of each protein within the sensu strictu species. The MAPKs Fus3 and Hog1 are the most conserved elements of the pathways, as indicated by the predominance of red in their matrices and the low N/S values. Pbs2 is found in all the species, whereas Ste7 (a Pbs2 duplicate) is present only in yeast, as indicated by the completely black columns and rows in its matrix, corresponding to the higher eukaryotes. The remaining proteins in a only have orthologues in yeast.

Evolutionary history of MAPKs and their interacting partners. (a) The phylogenetic tree of 15 yeast species and 7 higher eukaryote species used in deducing gene duplication events. Each speciation event (S₀,S₁,S₂,S₃,S₄…S₉) leads to the introduction of a new or duplicate gene in the upper branch. (b) MAPK-interacting partners from Fig. 1a, which are introduced de novo into the yeast lineage, are shown alongside the speciation event that created them. (c) Gene duplications accounting for all the remaining interacting partners not in b. Many of these have the new genes in b as their parent. (d) The only two MAPK-interacting partners, Pbs2 and Rlm1, found in all the species have changed their specificities towards MAPKs during evolution. In the yeast, the orthologue of the downstream transcription factor Rlm1 is activated by the orthologue of Slt2 (the MAPK of the hypo-osmolar pathway). In the higher eukaryotes, however, (for example human, mouse and rat), the Rlm1 orthologue (MEF2A) is activated by the Hog1 orthologue (p38α, refs 18, 25). A similar change occurs upstream. In yeast, the Pbs2 orthologue activates the Hog1 orthologue, whereas in human, mouse and rat the Pbs2 orthologue (MEK1/2) activates the Fus3 orthologue (ERK1/2, ref. 13). Thus despite being highly conserved, the MAPKs show flexibility in acquiring and swapping interaction partners through evolution.

Sequence analysis of MAPKs Fus3 and Hog1. (a) The degree of variability, as measured from entropy, in a multiple sequence alignment of four MAPKs and their orthologues from 22 species (Supplementary Information, Data Analysis) is shown on the structure of Fus3. High entropy (red) positions are constrained to the surface, whereas low entropy (blue) positions are in the interior. (b) Residues on Fus3 deduced computationally as putatively being responsible for the differences in Fus3 and Hog1 specificities are shown highlighted on its steric surface. The protein structure on the left has the same orientation as that in a, and the structure on the right has been rotated 180° about the vertical axis. The residues are coloured according to the segment they belong to, with the same colours as used in the six segments in c and d. Some residues, such as P80, F83, E84 and W348 shown on the structure on the right, distinguish themselves by being present on Fus3 and not on Hog1. (c, d) The segments A/a, B/b, C/c, D/d, E/e and F/f in Fus3/Hog1 used to build the hybrid kinases are shown on the structures of Fus3 and p38α (mouse Hog1 orthologue), respectively. Five regions linking these segments, shown in blue, were chosen on the basis of their strict conservation in all the MAPKs in the multiple sequence alignment used in a. Specific residues previously identified in the literature are highlighted on both structures (Supplementary Information, Section 2). Most of these residues are common to both of the MAPKs and their orthologues from other species; however, they are shown highlighted on one or the other to reflect the MAPK in which they were identified. Thus, except for D112 and H113, these residues cannot be important for specificity.

Several hybrid MAPKs function in vivo to faithfully transduce and cross-wire the pheromone and hyper-osmolar signals. (a) The mean fold change in pFUS1 activity (upper panel, measured by a fluorescent reporter) in ~100 induvidual fus3Δ kss1Δ haploid cells containing the hybrid MAPKs expressed from a low-copy plasmid, after stimulation for 2 h by 0.6 μM pheromone or 1 M sorbitol. The percentage of residues in the hybrid that belong to Fus3 and differ from Hog1 is shown in parentheses. Serial dilutions show the mating efficiency of fus3Δ kss1Δ MATa haploids containing the indicated hybrids. Error bars reflect the intrinsically large range of response in the cells and not the measurement of uncertainty in a single cell. One hybrid expressed in low-copy plasmid (upper panel), and two hybrids expressed in high-copy plasmid (lower panel) mediate a cross-wiring whereby sorbitol evokes pFUS1 activity. The persistence of this cross-wiring after deleting STE7 is indicated by ‘Δ’. Hybrids showing constitutive pFUS1 activity are marked with an asterisk. Sorbitol-induced pFUS1 activity of aBcdeF was measured in fus3Δkss1Δhog1Δ cells. (b) The mean fold change in pSTL1 activity measuring an osmolar response, in ~100 single hog1Δ haploid cells containing the hybrid MAPKs and stimulated in the same manner as in a. The axes for fold change are scaled differently for the pheromone and sorbitol stimuli. Serial dilutions show the efficient growth of hog1Δ strains carrying the hybrid on plates containing 1 M sorbitol. Two hybrids mediated a cross-wiring showing pSTL1 activity on pheromone exposure (Supplementary Information, Data Analysis). The pheromone induced pSTL1 activity of aBcdEF was measured in fus3Δ kss1Δ cells.

Modular design allows some hybrid MAPKs to be activated by either input and others capable of activating either output. The protein space between extant Fus3 and Hog1 is rich in function. (a, b) Differential interference contrast (DIC) and fluorescence images of cells containing three hybrids: aBcdeF and AbCdEf on a low-copy plasmid and ABcdEF on a high-copy plasmid. Cells were stimulated with pheromone (left) and sorbitol (right). Green fluorescence (a) shows pFUS1 activity in fus3Δ kss1Δ cells (which, except for aBcdeF, have an intact osmolar pathway) mediated by the hybrid on pheromone or sorbitol stimulation. Red fluorescence (b) shows the pSTL1 activity in hog1Δ cells (which have an intact pheromone pathway) mediated by the hybrid on stimulation by pheromone or sorbitol. (c) aBcdeF interacts upstream only with the MAPKK Pbs2 of the osmolar pathway, but can interact downstream with transcription factors of both pathways (Ste12, Hot1). Thus sufficiently non-overlapping groups of residues on the original MAPKs must be responsible for these downstream interactions. Analogously, AbCdEf interacts downstream only with Hot1 but interacts upstream with the MAPKKs Ste7 and Pbs2. Therefore sufficiently non-overlapping groups of residues are also responsible for these upstream interactions. (d) Functional properties of each hybrid are summarized schematically in a 2 × 2 matrix. The hybrids are arranged from Hog1 (top) to Fus3 (bottom). Hybrids in each successive row below Hog1 have one more segment taken from Fus3. The hybrids discussed in a, b and c are underlined. Each element of the matrix denotes the hybrids ability to mediate one of four distinct input-output characteristics in a cell. Top left (green) indicates pheromone response to pheromone stimulus. Top right (green) indicates pheromone response to sorbitol stimulus. Bottom left (red) indicates sorbitol response to pheromone stimulus. Bottom right (red) indicates sorbitol response to sorbitol stimulus. The bottom right is grey for three hybrids rescuing growth on 1 M sorbitol plates without showing a transcriptional response (Supplementary Information, Fig. S4b, c). Our sampling of the intervening MAPK protein space between Hog1 and Fus3 reveals several continuous paths via functioning intermediates.