ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.Energy-dependent proteolysis is responsible for more than 90% of the protein turnover inside the cell (1). This process both removes misfolded and aggregated proteins as part of the response of the cell to stress and controls the concentrations of regulatory proteins (2, 3). In prokaryotes and eukaryotic organelles, energy-dependent proteases fall into five classes as follows: ClpAP, ClpXP, Lon, HslUV (also referred to as ClpYQ), and HflB (also referred to as FtsH). In Archaea, analogous functions are performed by the archaebacterial proteasome, consisting of the proteasome-activating nucleotidase (PAN),³ working with the 20 S proteasome (4); in the cytoplasm and nucleus of eukaryotes, these same functions are performed by the 26 S proteasome (5). These different proteases show little sequence conservation outside the ATP-binding domains, but they share their overall architecture. They all form oligomeric, barrel-shaped complexes composed of one or more rings with the active sites of proteolysis sequestered inside a central degradation chamber (6). Access channels to these sites are narrow, and proteins have to be unfolded to gain entry (6). Regulatory particles belonging to the AAA family of molecular chaperones assemble on either end of the proteolytic chamber and recognize substrates destined for degradation. After recognition, the regulatory particles translocate the substrate through a central channel to the proteolytic chamber and in doing so unravel folded domains within the substrate. Translocation and unfolding are driven by ATP hydrolysis by the regulatory particles, with conformational changes in the protease transmitted to the substrate by conserved residues in the loops lining the channel (7–10).Protein degradation by AAA proteases is tightly regulated. Most proteins are targeted to ClpAP, ClpXP, HslUV, Lon, HflB, and PAN by sequence motifs in their primary structure (11–17). Sometimes adaptor proteins recognize and bind sequence elements in substrates and deliver them to the protease, and other times the protease recognizes sequence elements directly (18, 19). In contrast, proteins are typically targeted to the 26 S proteasome through the covalent attachment of polyubiquitin chains (20). Thus, substrates appear to be selected for degradation based on the presence of specific recognition elements in the protein substrates.However, other mechanisms may also affect the specificity of degradation by prokaryotic proteases. Individual proteases recognize a wide range of targeting signals (11, 16). (For example, Escherichia coli ClpXP recognizes sequences belonging to five distinct classes of consensus sequences (11), and ClpAP, Lon, and FtsH can bind to unstructured regions in proteins with a wide range of amino acid sequences (21–23).) One illustration of the loose specificity in targeting signals is the ability of a mitochondrial presequence to target proteins to the proteases ClpAP (24) and HslUV in vitro (see below). In addition, substrates are commonly acted upon by several different proteases in E. coli. For instance, proteins containing the 11-residue ssrA peptide at their C termini can be recognized by ClpAP, ClpXP, FtsH, Lon, and the archaebacterial proteasome (4, 25–27). Similarly, some substrates of Lon can be degraded by HslUV in vivo (28).It is not clear how degradation remains selective despite the loose specificity of targeting signals. We propose that the intrinsic protein unfolding ability of AAA proteases and the stabilities of substrates against unfolding play a role in determining the fate of cellular proteins. For example, ClpXP releases hard-to-unfold substrates when it encounters them and degrades destabilized titin variants 20-fold faster than wild type titin (29). The membrane-bound AAA protease FtsH has a weak unfolding ability, which allows this protease to act selectively on damaged and unfolded polypeptides (30). Here we find that the relative unfolding abilities of ATP-dependent proteases vary more than 100-fold and that the unfolding abilities of proteases belonging to the same class but originating from different species appear to be conserved. The unfolding abilities also seem to be intrinsic properties of the proteases themselves rather than other cytosolic factors, such as chaperones. Differences in protease unfolding abilities may contribute to substrate selectivity during protein degradation. For example, expression of a protease with a weak unfolding ability during a stress response could allow the selective elimination of unfolded, misfolded, or otherwise aberrant proteins and spare stable proteins from destruction (30).