A Gatekeeper Residue of ClpS1 from Arabidopsis thaliana Chloroplasts Determines its Affinity Towards Substrates of the Bacterial N-End Rule.

Proteins that are to be eliminated must be proficiently recognized by proteolytic systems so that inadvertent elimination of useful proteins is avoided. One mechanism to ensure proper recognition is the presence of N-terminal degradation signals (N-degrons) that are targeted by adaptor proteins (N-recognins). The members of the caseinolytic protease S (ClpS) family of N-recognins identify targets bearing an N-terminal phenylalanine, tyrosine, tryptophan or leucine residue, and then present them to a protease system. This process is known as the 'bacterial N-end rule'. The presence of a ClpS protein in Arabidopsis thaliana chloroplasts (AtClpS1) prompted the hypothesis that the bacterial N-end rule exists in this organelle. However, the specificity of AtClpS1 is unknown. Here we show that AtClpS1 has the ability to recognize bacterial N-degrons, albeit with low affinity. Recognition was assessed by the effect of purified AtClpS1 on the degradation of fluorescent variants bearing bacterial N-degrons. In many bacterial ClpS proteins, a methionine residue acts as a 'gatekeeper' residue, fine-tuning the specificity of the N-recognin. In plants, the amino acid at that position is an arginine. Replacement of this arginine for methionine in recombinant AtClpS1 allows for high-affinity binding to classical N-degrons of the bacterial N-end rule, suggesting that the arginine residue in the substrate-binding site may also act as a gatekeeper for plant substrates.

Characterization of the accessory protein ClpT1 from Arabidopsis thaliana: oligomerization status and interaction with Hsp100 chaperones.

BACKGROUND: The caseinolytic protease (Clp) is crucial for chloroplast biogenesis and proteostasis. The Arabidopsis Clp consists of two heptameric rings (P and R rings) assembled from nine distinct subunits. Hsp100 chaperones (ClpC1/2 and ClpD) are believed to dock to the axial pores of Clp and then transfer unfolded polypeptides destined to degradation. The adaptor proteins ClpT1 and 2 attach to the protease, apparently blocking the chaperone binding sites. This competition was suggested to regulate Clp activity. Also, monomerization of ClpT1 from dimers in the stroma triggers P and R rings association. So, oligomerization status of ClpT1 seems to control the assembly of the Clp protease. RESULTS: In this work, ClpT1 was obtained in a recombinant form and purified. In solution, it mostly consists of monomers while dimers represent a small fraction of the population. Enrichment of the dimer fraction could only be achieved by stabilization with a crosslinker reagent. We demonstrate that ClpT1 specifically interacts with the Hsp100 chaperones ClpC2 and ClpD. In addition, ClpT1 stimulates the ATPase activity of ClpD by more than 50% when both are present in a 1:1 molar ratio. Outside this optimal proportion, the stimulatory effect of ClpT1 on the ATPase activity of ClpD declines. CONCLUSIONS: The accessory protein ClpT1 behaves as a monomer in solution. It interacts with the chloroplastic Hsp100 chaperones ClpC2 and ClpD and tightly modulates the ATPase activity of the latter. Our results provide new experimental evidence that may contribute to revise and expand the existing models that were proposed to explain the roles of this poorly understood regulatory protein.

Toward a unified model of the action of CLP/HSP100 chaperones in chloroplasts.

In chloroplasts, Hsp70 and Hsp100 chaperones have been long suspected to be the motors that provide the necessary energy for the import of precursor proteins destined to the organelle. The chaperones associate with the import translocon and meet the transit peptides as they emerge through the channel. After decades of active research, recent findings demonstrated that Hsp100 chaperones recognize transit peptides both in vitro and in vivo. Moreover, Hsp70 also plays a part in precursor import. The updated model of protein translocation into chloroplasts now presents new questions about the role of the chaperones in the process.