The contribution of individual domains of chloroplast protein AtDeg2 to its chaperone and proteolytic activities

Abstract

The thylakoid protease AtDeg2 is a non-ATP hydrolyzing chloroplast protease/chaperone peripherally connected with stromal side of thylakoid membrane. Its linear structure consists of protease domain and two PDZ domains. To unveil the significance of individual domains, chaperone and proteolytic activities of AtDeg2, its mutated recombinant versions have been developed and their ability to suppress protein aggregation and resolubilization of protein aggregates as well as the ability to degrade substrate protein was examined in vitro. Our work reveals for the first time that AtDeg2 is able not only to suppress aggregation of denatured proteins, but to resolubilize existing protein aggregates as well. We show that PDZ2 domain contributes significantly to both chaperone and protease activities of AtDeg2, whereas PDZ1 is required for chaperone but superfluous for proteolytic activity. Protease domain – but not S-268 in its catalytic site – contributes to chaperone activities of AtDeg2. These results show an entirely new function of AtDeg2 chaperone/protease (i.e., disaggregation of protein aggregates) and allow to identify structural motifs required for “old” and new functions of AtDeg2.

Chloroplast protease/chaperone AtDeg2 influences cotyledons opening and reproductive development in Arabidopsis

“…Besides being a protease, AtDeg2 also has a chaperone activity. Specifically, this protein is able to inhibit the aggregation of denatured lysozyme in vitro [5,6] and can cause in vitro disaggregation of pre-existing aggregates of denatured lysozyme [6]. We have shown that the PDZ2 domain is required both for the chaperone and protease activities of AtDeg2, whereas PDZ1 contributes to the chaperone but not to the protease activity of AtDeg2.…”

“…We have shown that the PDZ2 domain is required both for the chaperone and protease activities of AtDeg2, whereas PDZ1 contributes to the chaperone but not to the protease activity of AtDeg2. The protease domain -but not S268 in its catalytic center -was demonstrated to contribute to AtDeg2 chaperone activity [6]. AtDeg1, another non-ATP hydrolyzing chloroplast protease that belongs to the Deg group, was shown to have a chaperone (refoldase) activity as well, which probably requires protease domain because substitution of the proteolytically active serine residue (S280) by the proteolytically inactive alanine residue (A) considerably reduced the refoldase activity of AtDeg1.…”

“…-GFP) that lacked chaperone activity but was proteolytically active [6], or AtDeg2 with a C-terminal GFP tag that lacked its proteolytic activity but still had its chaperone function (35S:AtDEG2…”

“…-GFP) [6]. To make the results plausible, the data concerning phenotypic traits were collected so that the mutants, the transformants, and WT plants represented identical ontogenetic stages, rather than identical age, as had been reported previously [9].…”

“…It belongs to the S1C subfamily of the S1 family (chymotrypsin A, Bos taurus) and is classified in the Merops database as "Deg2 chloroplast peptidase (Arabidopsis thaliana)". Recombinant AtDeg2 was shown to catalyze the in vitro hydrolysis of various artificial protein substrates including gelatin [4], β-casein [5,6], and fluorescence-labeled casein [7] indicating that AtDeg2 is a bona fide proteolytic enzyme. The mature AtDeg2 molecule…”