RESUMO
The adaptor protein ClpS associates to the Clp protease and promotes degradation of N-end rule substrates in eubacteria and in algal/plant chloroplasts. Cyanobacteria are unusual in having two distinct ClpS paralogs. Although ClpSl is typical of bacterial ClpS, ClpS2 differs in crucial ways. ClpS2 in Synechococcus elongatus is a relatively low-abundant, soluble protein essential for phototrophic growth. Like ClpSl, ClpS2 binds to the ClpCP3/R protease to block α-casein degradation and promote that of N-end rule substrates in vitro. However, their substrate specificity differs, with ClpSl recognizing destabilizing Phe and Tyr residues at the substrate N-terminus whereas ClpS2 recognizes Leu. Overall, ClpS2 appears to have independently evolved in cyanobacteria to degrade a particular group of proteins, whose turnover is vital for cell viability.
Assuntos
Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Synechococcus/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência Conservada , Cinética , Leucina/metabolismo , Viabilidade Microbiana , Dados de Sequência Molecular , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Filogenia , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , Synechococcus/crescimento & desenvolvimentoRESUMO
The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.
