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Identification of major lysine residues of S 3 ‐RNase of Petunia inflata involved in ubiquitin–26S proteasome‐mediated degradation in vitro
Author(s) -
Hua Zhihua,
Kao Tehhui
Publication year - 2008
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2008.03487.x
Subject(s) - biology , rnase p , rnase mrp , pollen tube , proteasome , mutant , ubiquitin , f box protein , biochemistry , ribonuclease , protein degradation , rnase h , microbiology and biotechnology , ubiquitin ligase , gene , pollen , rna , botany , pollination
Summary S‐RNase‐based self‐incompatibility has been identified in three flowering plant families, including the Solanaceae, and this self/non‐self recognition mechanism between pollen and pistil is controlled by two polymorphic genes at the S ‐locus, S‐RNase and S‐locus F‐box ( SLF ). S‐RNase is produced in the pistil and taken up by pollen tubes in a non‐ S‐ haplotype‐specific manner. How an allelic product of SLF interacts with self and non‐self S‐RNases to result in growth inhibition of self pollen tubes is not completely understood. One model predicts that SLF targets non‐self S‐RNases for ubiquitin/26S proteasome‐mediated degradation, thereby only allowing self S‐RNase to exert cytotoxic activity inside a pollen tube. To test this model, we studied whether any of the 20 lysine residues in S 3 ‐RNase of Petunia inflata might be targets for ubiquitination. We identified six lysines near the C‐terminus for which mutation to arginine significantly reduced ubiquitination and degradation of the mutant S 3 ‐RNase, GST:S 3 ‐RNase (K141–164R) in pollen tube extracts. We further showed that GST:S 3 ‐RNase (K141–164R) and GST:S 3 ‐RNase had similar RNase activity, suggesting that their degradation was probably not caused by an ER‐associated protein degradation pathway that removes mis‐folded proteins. Finally, we showed that PiSBP1 ( P. inflata S‐RNase binding protein 1), a potential RING‐HC subunit of the PiSLF ( P. inflata SLF)‐containing E3‐like complex, could target S‐RNase for ubiquitination in vitro . All these results suggest that ubiquitin/26S proteasome‐dependent degradation of S‐RNase may be an integral part of the S‐RNase‐based self‐incompatibility mechanism.