N,N'-dicyclohexylcarbodiimide cross-linking suggests a central core of helices 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 only in maize carrying the cms-T cytoplasm. URF13 is associated with cytoplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 is predicted to contain three transmembrane alpha-helices and is a receptor for the pathotoxins (T-toxins) produced by B. maydis race T and P. maydis. Binding of T-toxin to URF13 leads to membrane permeability. Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mitochondria from the permeability caused by T-toxin or methomyl. Using mutated forms of URF13 expressed in E. coli cells, we determined the molecular 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-expressing cells did not protect the cells from permeability caused by T-toxin or methomyl. DCCD cross-linking was greatly reduced in K37I-URF13 and in D39V-URF13-expressing cells, but it was unaffected in K32A-URF13-expressing cells. Binding of methomyl or T-toxin decreases DCCD cross-linking of URF13 oligomers expressed in either E. coli or cms-T mitochondria. We conclude that Asp-39 in helix II is cross-linked by DCCD to Lys-37 in helix II of an adjacent URF13 molecule and that this cross-linking protects against toxin-mediated permeabilization. Our results also indicate that helices II form a central core in URF13 oligomers.
Rhoads
D M
DM
Department of Botany, Duke University, Durham, NC 27708.
Kaspi
C I
CI
Levings
C S
CS
Siedow
J N
JN
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004024:Dicyclohexylcarbodiimide; D004926:Escherichia coli / Q000378:metabolism; D005658:Fungi / Q000472:pathogenicity*; D008724:Methomyl / Q000633:toxicity; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D003904:Mitosporic Fungi / Q000472:pathogenicity; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000378:metabolism / Q000633:toxicity*; D010101:Oxygen Consumption / Q000187:drug effects; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D003313:Zea mays / Q000378:metabolism*
D003432:Cross-Linking Reagents; D017931:DNA Primers; D024101:Mitochondrial Proteins; D009183:Mycotoxins; D010940:Plant Proteins; D011994:Recombinant Proteins; C071393:URF13 protein, Zea mays; D008724:Methomyl; D004024:Dicyclohexylcarbodiimide