Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability

Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the int eractions between the different proteins, we used the two-hybrid system, wh ich showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRI II, without an arginine requirement. To define the interacting domains of A rgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA- binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm 1. The putative alpha helix present in the MADS-box domain of ArgRI and Mcm 1 is their major region of interaction with ArgRIII. Interactions between t he two MADS-box proteins and ArgRIII were confirmed using affinity chromato graphy, The requirement for ArgRIII in the control of arginine metabolism c an be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed w ith the 'arginine boxes', We show that the impairment of arginine regulatio n in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1, A mutation in ArgRI, impairing its interaction with ArgRIII, lea ds to an unstable ArgRI protein in a wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate s ome physiological defects observed in an argRIII deletant strain.