Membrane Protein Degradation by FtsH Can Be Initiated from Either End

Abstract

FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.Energy-dependent proteolysis plays important roles in controlling and maintaining cellular functions. Whereas the 26S proteasome is a key player of these events in eukaryotic cells, HslUV, ClpAP, ClpXP, Lon, and FtsH and their homologs are known as ATP-dependent proteases in bacteria (13,23,38) as well as in the mitochondria and the chloroplasts (1, 23). The ATPase domains or subunits of these proteases belong to the AAAϩ ATPase family (29,34,38). Many of them are known to form ring-like assemblies of subunits (31, 43). The proteolytic active sites of these enzymes are thought to be located in the interior cavity of the assemblies; experimentally this has been shown for the proteasome, HslUV, ClpAP, and ClpXP (8,15,26,44). For the substrate peptide bond to be hydrolyzed, it must be presented to the catalytic center within the cavity. This implies that the substrate protein itself must be unfolded and translocated into the catalytic chamber (18,33,35). Presumably, these proteases utilize their ATPase functions to facilitate unfolding and translocation of substrate proteins.A characteristic feature of the energy-dependent proteolytic reactions is that they occur in processive manners (24). Once the enzyme initiates the reaction, it continues to work along the polypeptide chain to hydrolyze the substrate successively. Crucial questions here include (i) how the enzyme recognizes the initiation site on the substrate, (ii) how it initiates unfolding or translocation of the substrate, and (iii) how it continues the reaction along the polypeptide chain, expelling the product small peptides from the catalytic chamber. Studies on the Clp proteases demonstrated that these enzymes can recognize either the N-or C-terminal end of substrate polypeptides for the initiation of unfolding or proteolysis (12,14,17).Among the ATP-dependent proteases i...

AtFtsH6 is involved in the degradation of the light-harvesting complex II during high-light acclimation and senescence

“…Some of these protease activities may cooperate with FtsH6 in the degradation of Lhcb1 and Lhcb3. In E. coli, FtsH was found to be able to degrade both soluble and membrane proteins, initiating proteolysis at either the N terminus or C terminus (47). The substrate must be partially unfolded, because FtsH lacks a robust unfoldase activity (48).…”

The Thylakoid Lumen Protease Deg1 Is Involved in the Repair of Photosystem II from Photoinhibition in Arabidopsis

“…The role of these two proteases is consistent with their localization to the stromal side of the thylakoid membrane (Lindahl et al, 1996;Haussuhl et al, 2001). Deg2, or other stromal peptidases, cleave at the large stromal DE-loop ( Figure 7), creating new termini on this side of the membrane that can be recognized by FtsH, which in E. coli can degrade its substrates processively from either their N or C terminus (Chiba et al, 2002). The creation of two new termini on the stromal side of the membrane, in addition to the endogenous N terminus, is likely to enhance the rate of D1 degradation because these can serve as additional sites for the initiation of proteolysis.…”

Reconstitution of Membrane Proteolysis by FtsH

“…FtsH is also able to recognize a C-terminal tail for initiation of membrane protein degradation (16). For our further understanding of the molecular mechanisms of polypeptide extraction from the membrane and presentation to the proteolytic active site, it is essential to develop an in vitro system, in which membrane protein degradation by FtsH can be studied using membrane-integrated enzyme and substrates.…”