Clp/Hsp100 proteins are a class of hexameric molecular chaperones from the ever-growing family of AAA + (A
ssociated with diverse cellular a
ctivities) ATPases. They possess a wide range of functional roles, including protein folding assistance, protein degradation, disaggregation of denatured polypeptides and assembly of large molecular complexes [1
]. Most (if not all) Clp/Hsp100 proteins act as independent chaperones but some associate with oligomeric barrel-shaped proteases to degrade irreversibly damaged proteins [4
]. Notable examples are ClpA and ClpX from Escherichia coli
and ClpC from Bacillus subtilis
and Synechococcus elongatus
, which couple to their respective ClpP protease [6
In plants, the diversity of the Clp family is greater than in any other organism. The model plant A. thaliana
has at least 23 members [9
]. Hsp100 chaperones from the ClpX family (ClpX1-3) are found in mitochondria while those from the ClpC (ClpC1 and ClpC2) and ClpD subtype are chloroplastic. The ClpPR proteolytic core is represented by six different proteins (ClpP1-6) and four ClpR proteins (ClpR1-4). Three modulator/adaptor proteins (ClpT1, ClpT2 and ClpS) are thought to regulate substrate recognition and binding.
Recombinant AtClpC2 and AtClpD have been recently characterized [10
]. AtClpD was consistently obtained as two forms, one being the full-length protein and the other corresponding to a C-terminus-processed variant. The proteins displayed ATPase and foldase activity and self-assembled into hexamers. Clp/Hsp100 chaperones are constitutively expressed and are highly conserved among different plant species [11
]. Knockout plants in the Atclpc1
gene are pale green and show retarded growth, while Atclpc1
double mutants are not viable [12
]. All this evidence suggests that chloroplastic Clp proteins are key members in the process of protein quality control in the chloroplast.
In addition to their proposed housekeeping duties, ClpC proteins from plants have been implicated in the import of nuclear-encoded proteins into chloroplasts. Proteins destined to the organelle are synthesized as higher molecular mass precursors containing an N-terminal extension called transit peptide (TP). It acts as a signal that is recognized by the translocon channel, a bipartite complex with central pores through which precursors penetrate into the plastid stroma [13
]. A fraction of ClpC is localized in the stromal side of the chloroplast inner membrane and interacts with the import machinery when a precursor is being translocated [10
]. In A. thaliana
, chloroplasts isolated from insertional mutants in the clpc1
gene show a diminished rate of import [12
]. The current model of protein import into chloroplasts place Hsp100 chaperones (specifically, members of the ClpC subfamily) as motors pulling precursors by their TPs. Yet many aspects of this scenario are still to be elucidated, such as TP recognition by the chaperone, threading of the polypeptide through the Hsp100 ring and interplay with the Hsp70 network, which was also shown to interact with precursors [18
The step of substrate recognition has been studied in ClpA from E. coli
. This chaperone can recognize tags located at the N- or C-terminal end (e.g., the RepA tag and the SsrA tag, respectively) [19
]. Though the amino acid composition of these tags is well characterized, consensus sequences are still not defined, as many other untagged proteins are substrates of ClpA [21
]. It is thought that short exposed segments with hydrophobic amino acids and little (if any) tridimensional structure are recognized, like is the case for the substrates of Lon protease and the chaperones GroEL, DnaK, Trigger Factor and many others [22
]. However, it is not known which specific attributes are recognized by the Hsp100 chaperones associated with the translocation channel. Furthermore, since only a handful of Hsp100 targets have been identified, no obvious sequence pattern is available to analyze if a given TP could be an Hsp100 substrate.
The aim of this work was to elucidate some aspects of the process of TP recognition by Clp/Hsp100 chaperones. Several fusion proteins were constructed in which TPs were placed at different positions in a polypeptide. Then, binding to these probes by purified recombinant AtClpC2 and AtClpD was analyzed. Using this in vitro system, we found that AtClpC2 and AtClpD only interacted with the fusions that have the TPs in the natural location (i.e., at the N-terminus).