Differential tolerance for SEA domain misfolding encodes a MAPK pathway-specific response

Signaling pathways often share components yet produce highly specialized biological responses. How signaling specificity is achieved between pathways utilizing common components is a fundamental question. In budding yeast, the same transmembrane mucin, Msb2, regulates two Mitogen-Activated Protein Kinase (MAPK) pathways controlling filamentous growth (fMAPK) and the response to osmotic stress (HOG). How this shared sensor distinguishes between stimuli and regulates different pathways is not clear. Using structure-guided analysis, we identified a conserved SEA (Sea urchin sperm protein, Enterokinase, Agrin) domain in fungal mucins and found that mutations disrupting protein folding selectively impair one pathway (fMAPK) but were tolerated by another (HOG). Mechanistically, these differences revealed distinct modes of signal transmission. The fMAPK pathway required an intact SEA domain and the cytosolic tail, consistent with a cis signaling mechanism that required structural coupling across the membrane. In contrast, the HOG pathway functioned independently of the cytosolic tail and tolerated misfolded SEA domain variants, consistent with trans signaling mediated by extracellular domains of interacting partners. The HOG pathway may detect misfolding as part of its sensing mechanism, as stressors that induce protein misfolding required Msb2 for survival. This work reveals how differential tolerance to protein deformation confers signaling specificity and identifies sensor deformation as a general feature of mechanosensory pathways that respond to environmental stress. HIGHLIGHTSO_LISignaling pathways differ in tolerance to misfolding of a sensory domain C_LIO_LIMisfolded SEA domains retain function in a stress pathway (HOG) pathway but not a cell differentiation pathway (fMAPK) O_LIMisfolded SEA domain variants showed altered protein levels, mis-localization in the secretory pathway, and turnover by ERAD C_LIO_LINon-functional variants lacked residues that stabilize the structure through intramolecular bonds C_LI C_LIO_LIDifferential tolerance for misfolding revealed distinct modes of signaling O_LITrans signaling predominated in the HOG pathway and did not require proper SEA domain folding or the mucin cytosolic tail O_LIA dominant hyperactive variant next to the SEA domain revealed basal interactions with the CR domain of tetraspanin C_LIO_LIAlphaFold modeling showed distinct interactions occur between the SEA domain and tetraspanin in the basal and activated states C_LI C_LIO_LICis signaling predominated in the fMAPK pathway O_LIRequired a properly folded SEA domain and conformational coupling to the cytosolic tail C_LIO_LIYapsin processing was required for SEA domain activation and turnover of the mucin cytosolic tail C_LI C_LI C_LIO_LIHOG pathway may sense protein misfolding as part of its activation mechanism. C_LIO_LISEA domains are conserved throughout fungal mucins and mammalian glycoprotein sensors suggesting a generalizable mechanism C_LIO_LIProtein deformation may provide information to survival pathways about environmental stress. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/723240v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1cd30f3org.highwire.dtl.DTLVardef@48c96corg.highwire.dtl.DTLVardef@9fffc2org.highwire.dtl.DTLVardef@504b1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Signaling pathways often share components yet activate different effector processes through mechanisms that remain unclear. The same mucin regulates two MAPK pathways (red and green), and the discovery of a conserved SEA domain provided insights into specificity mechanisms. In the fMAPK pathway that regulates filamentous growth, the mucin works in a classical manner, where an external signal (in this case underglycosylation by glucose limitation) transduces a signal to the cytosolic domain in cis. By comparison, the HOG pathway that responds to osmotic stress displayed a remarkable tolerance for mucin and SEA domain deformation. Protein variants that caused SEA domain misfolding, mislocalization, and degradation by ERAD retained function in the HOG pathway. Truncations that removed the cytosolic tail and transmembrane anchor were also functional. These phenotypes support a trans activation mechanism with external partners that was preferential for activation of the HOG pathway. SEA domain deformation may be induced by environmental stress as a trigger for the HOG pathway. Cells may detect misfolding of protein domains to gain information about environmental stress.