NN'-DICYCLOHEXYLCARBODIIMIDE CROSS-LINKING SUGGESTS A CENTRAL CORE OFHELICES-II IN OLIGOMERS OF URF13, THE PORE-FORMING T-TOXIN RECEPTOR OF CMS-T MAIZE MITOCHONDRIA

URF13 is a mitochondrially encoded, integral membrane protein found on ly in maize carrying the cms-T cytoplasm. URF13 is associated with cyt oplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF1 3 is predicted to contain three transmembrane alpha-helices and is a r eceptor for the pathotoxins (T-toxins) produced by B. maydis race T an d P. maydis. Binding of T-toxin to URF13 leads to membrane permeabilit y. Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mit ochondria from the permeability caused by T-toxin of methomyl. Using m utated forms of URF13 expressed in E. coli cells, we determined the mo lecular mechanism of DCCD protection. We separately changed Lys-37 in helix II to isoleucine (K37I-URF13) and Lys-32 in the helix I/helix II loop region to alanine (K32A-URF13). DCCD treatment of K37I-URF13-exp ressing cells did not protect the cells from permeability caused by T- toxin or methomyl. DCCD cross-linking was greatly reduced in K37I-URF1 3 and in D39V-URF13-expressing cells, but it was unaffected in K32A-UR F13-expressing cells. Binding of methomyl or T-toxin decreases DCCD cr oss-linking of URF13 oligomers expressed in either E. coli or cms-T mi tochondria. We conclude that Asp-39 in helix II is cross-linked by DCC D to Lys-37 in helix II of an adjacent URF13 molecule and that this cr oss-linking protects against toxin-mediated permeabilization. Our resu lts also indicate that helices II form a central core in URF13 oligome rs.