Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in Brassica self-incompatibility

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rlch protein), and S-locus glycoprotein (SLG). SP11, located in the pollen cost, determines pollen S-haplotype specificity, whereas SRK, located on the plesme membrane of stigmetic papille cells, determines stigmetic S-heplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K_d = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the atigmatic membrane forms e high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) locking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK end soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.