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
Dm. Rhoads et al., 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, Proceedings of the National Academy of Sciences of the United Statesof America, 91(17), 1994, pp. 8253-8257
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.