A Cytoplasmic Male Sterility-Associated Mitochondrial Peptide in Common Bean Is Post-Translationally Regulated

Abstract

Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-or f 239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome. The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally.

Proteomic Analysis of Rat Liver Peroxisome

“…In eukaryotes these functions are affected by more complex proteosome machineries, but a Lon protease homolog has been found in mitochondria of yeast and mammals (32,33). Lon proteases in plants are now classified into three isoforms; Lon1, Lon2, and Lon3 (21). Sequence comparison revealed the relationship among various Lon isoforms (Fig.…”

“…A close examination of sequences of Lon proteases of various origins indicated that at least two isoforms exist in mammalian cells. One isoform has been characterized as mitochondrial Lon protease, which is homologous to plant Lon2 sequences (21,22). The present isoform is likely an orthologue of the plant Lon1.…”

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

“…The only reported activity of chloroplast DegP is the initial cleavage of the D1 protein of PSII during repair from photoinhibition . Information about activities of mitochondrial proteases is even more scarce, with the involvement of Lon protease in the control of cytoplasmic male sterility (Sarria et al, 1998) being the only report to date.…”

“…In Arabidopsis, the proteolytic machinery of chloroplasts consists primarily of three families of ATP-dependent proteases, Clp, Lon, and FtsH, and one family of ATPindependent proteases, DegP (for review, see Adam and Clarke, 2002;Sokolenko et al, 2002). Homologous enzymes are found also in mitochondria (Sarria et al, 1998;Adam et al, 2001;Halperin et al, 2001b;Kolodziejczak et al, 2002). All these families have well-characterized homologs in Escherichia coli (for review, see Clarke, 1999;Adam, 2000).…”

“…One isozyme, ClpP2, is found in mitochondria, where it may associate with the regulatory subunit ClpX (Halperin et al, 2001b;Peltier et al, 2004). The four Arabidopsis isomers of Lon protease are predicted to reside in either chloroplasts or mitochondria (Sarria et al, 1998;Adam et al, 2001;Sokolenko et al, 2002), as are the different forms of the FtsH protease. FtsH1, 2, and 5 were found as integral proteins in the thylakoid membrane, with their ATPbinding domain and catalytic zinc-binding site facing the stroma (Lindahl et al, 1996;Chen et al, 2000;Sakamoto et al, 2002).…”