it was previously reported that mcs4 physically interacts with the wis4 mapkkk of the spc1 mapk cascade . in addition , we recently discovered that wis4 and its paralog , win1 , form a stable heteromer complex . the following observations strongly suggest that the mcs4 rr is an integral component of the ternary complex with the wis4 and win1 mapkkks ( fig . 2a ) found that heteromer association between the wis4 and win1 mapkkks is not affected by stress treatments . third , disruption of the mcs4 gene ( mcs4 ) compromises the interaction between the mapkkks both in the presence and absence of stress ( fig .  2a , lanes 711 ) . ( a ) the mcs4 rr forms a stable complex with the wis4-win1 mapkkk heteromer . the wis1 mapkk and the tdh1 gapdh are also found on the complex , and the mpr1 hpt - mcs4rr association is detectable in the absence of stress . ( b ) phosphorelay through the two - component system transmits oxidative stress signals to the spc1 mapk cascade . oxidative stress enhances the tdh1-mcs4 association , which is critical for the interaction of the mpr1 hpt with the mcs4 rr and successful phosphorelay between them . ( c ) high osmolarity stress signals are transmitted to the mcs4-mapkkk complex by an unknown mechanism independent of the two - component system . tdh1 is released from mcs4 in response to osmostress . because tdh1 is required for the mpr1-mcs4 association , mpr1 is likely to dissociate from mcs4 upon osmostress . stress - induced activation of the mcs4-mapkkk complex results in phosphorylation and activation of the wis1 mapkk and its release from the complex . wis4 , win1 , wis1 , mcs4 and tdh1 were expressed from their own genomic loci with carboxyl terminal epitope tags or the ha epitope tag followed by the tandem affinity purification tag ( hatap ) or six consecutive histidine residues ( ha6h ) . cells in early log - phase were treated with 0.6 m kcl ( osmostress ) or 0.73 mm h2o2 ( oxidative stress ) for the indicated times . the cell lysate supernatant after high - speed centrifugation was subjected to the co - precipitation assay to monitor protein - protein interactions . ( a ) disruption of the mcs4 gene ( mcs4 ) weakens the wis4-win1 mapkkk heteromer association . win1-hatap was purified with igg - sepharose from lysate of cells co - expressing wis4-myc , and the proteins in the purified fractions were detected by immunoblotting . the levels of wis4-myc in the lysate used for the affinity purification are shown in the bottom panel . lane 1 , a strain expressing untagged win1 as a negative control ; lanes 26 , mcs4 cells ; lanes 711 , mcs4 cells . ( b ) the wis1 mapkk dissociates from the mapkkk complex in response to both phosphorelay - dependent and -independent signals . the win1-hatap was purified with igg - sepharose from lysate of cells co - expressing wis1-flag ( lanes 610 ) , and the proteins in the purified fractions were detected by immunoblotting . a strain expressing untagged win1 was used as a negative control ( lanes 15 ) . ( c ) the tdh1 gapdh physically associates with the mcs4 rr independently of the wis4 and win1 mapkkks . lysate of the double gene disruptant ( wis4 win1 ) expressing tdh1-ha6h and mcs4-myc was subjected to affinity - purification of tdh1 with ni - nta - agarose ( lanes 15 ) , and the proteins in the purified fractions were detected . a strain expressing untagged tdh1 was used as a negative control ( lane 6 ) . ( d ) tdh1 is detached from the mcs4 rr in response to osmotic stress . tdh1 was purified as described in ( c ) from a strain expressing mcs4-flag ( lanes 58 ) . a strain expressing untagged tdh1 was used as a negative control ( lanes 14 ) . the integrity of the mcs4 rr - mapkkk complex appears to be important for its physical interaction with the downstream wis1 mapkk , because the mapkkk - mapkk interaction is not detectable in strains lacking mcs4 or one of the mapkkks . thus , it is very likely that the mcs4 defect in osmostress - induced activation of the spc1 cascade is due to the compromised interaction between the mapkkks and the wis1 mapkk . the wis1 mapkk is released from the mcs4-mapkkk complex in response to phosphorelay - dependent ( h2o2 ) and -independent ( osmostress ) signals ( fig . while it remains unknown how osmostress is sensed and transmitted to the mapkkk complex , the peroxide stress sensor linked to the spc1 mapk cascade is the multi - step phosphorelay composed of the mak2/mak3 histidine kinases , the mpr1 hpt and the mcs4 rr ( fig . we previously reported that glyceraldehyde-3-phosphoate dehydrogenase ( gapdh ) , a glycolytic enzyme , is involved in the mcs4-mediated , peroxide signaling in fission yeast . disruption of the tdh1 gene encoding gapdh ( tdh1 ) significantly compromises the physical association between the mcs4 rr and mpr1 hpt , suggesting that the tdh1 gapdh promotes phosphorelay signaling to the mcs4 rr . as a consequence , the tdh1 mutant is defective in activation of the spc1 mapk by peroxide stress but not other forms of stress . although we initially identified tdh1 as a protein co - purified with the wis4 mapkkk and mcs4 , association of tdh1 to mcs4 is detectable even in the strain lacking both wis4 and win1 mapkkks ( fig . consistent with its role in peroxide stress signaling , binding of tdh1 to the mcs4 rr is enhanced after h2o2-treatment . intriguingly , we found that the tdh1 gapdh promptly dissociates from the mcs4 rr in response to high osmolarity stress ( fig . because tdh1 is required only for peroxide signaling to the mapk cascade , detachment of tdh1 from the mcs4-mapkkk complex might represent conversion of the complex to the osmosignaling form ( fig . our studies garnered evidence for protein - protein interactions that build a multi - protein complex assembled around the wis4-win1 mapkkk heteromer ( fig .  1 ) . we are beginning to understand the mechanics of this elaborate apparatus that mediates activation of the s. pombe spc1 mapk cascade in response to different stress stimuli . of particular note is the dual role of the mcs4 rr in the mapkkk complex . oxidative stress stimuli sensed by the mak histidine kinases initiate phosphorelay to the mcs4 rr via the mpr1 hpt , and the phosphorylation of mcs4 triggers activation of the mapkkk complex ( fig . 1b ) . on the other hand , mcs4 is essential for the integrity of the mapkkk complex , and the loss of mcs4 destabilizes the wis4-win1 heteromer association , which appears to be important for the mapkkk - mapkk interaction . unexpectedly , the glycolytic enzyme gapdh may serve as a modulator of the mcs4 function . the tdh1 gapdh bound to mcs4 promotes physical interaction between the mpr1 hpt and mcs4 rr , thus accelerating the phosphorelay to transmit oxidative stress signals . like in the tdh1 mutant , dissociation of tdh1 from mcs4 upon osmostress ( fig . 2d ) may destabilize the mpr1-mcs4 interaction and convert the mapkkk complex to a configuration dedicated for osmostress signaling ( fig . no osmostress sensor has been identified in fission yeast , and it remains to be discovered how the stress signals are transmitted to the mapkkk complex . although the mcs4 rr may not be the receiver of osmostress signals within the complex , it is conceivable that the mcs4-mapkkk interaction plays similar , pivotal roles in both osmostress- and oxidative stress - induced activation of the mapkkks .
in a  two - component system ,  extracellular stimuli are transmitted by the transfer of a phosphoryl group from a sensor histidine kinase to a response regulator ( rr ) , a mechanism referred to as phosphorelay . in the fission yeast schizosaccharomyces 
 pombe , peroxide stress signals are transmitted by phosphorelay to the mcs4 rr , which activates the spc1 map kinase ( mapk ) cascade . 
 we previously demonstrated that a glycolytic enzyme , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) physically interacts with mcs4 and promotes phosphorelay signaling to mcs4 . 
 independently of the phosphorelay mechanism , mcs4 also plays a critical role in osmostress signaling , as a part of the stable ternary complex with the wis4 and win1 mapk kinase kinases ( mapkkks ) . 
 interestingly , gapdh dissociates from mcs4 upon osmostress , while oxidative stress promotes their association . 
 the mcs4 rr may serve as a switching hub that mediates activation of the wis4-win1 mapkkk heteromer in response to different forms of stress .