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Genes to Cells (2006) 11, 261-268. doi:10.1111/j.1365-2443.2006.00940.x
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An AAA protease FtsH can initiate proteolysis from internal sites of a model substrate, apo-flavodoxin

Takashi Okunoa, Kunitoshi Yamanaka and Teru Ogura*

Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Escherichia coli FtsH, which belongs to the AAA (ATPases associated with diverse cellular activities) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. It has been proposed that ATP-dependent proteases unfold substrate proteins and initiate a processive proteolysis from either terminus of the substrate polypeptide. We have found that FtsH degrades E. coli apo-flavodoxin (apo-Fld) but not holo-Fld containing non-covalently bound flavin mononucleotide (FMN). A mutant Fld carrying a substitution of Tyr94 to Asp (FldYD) with a lower affinity for FMN was efficiently degraded by FtsH. To elucidate the directionality of FldYD degradation by FtsH, we constructed several FldYD fusion proteins with glutathione S-transferase (GST), green fluorescent protein (GFP), or both GST and GFP. It was found that FtsH was able to initiate degradation of the FldYD moiety even when it was sandwiched by GST and GFP. Evidence indicated that FtsH can initiate proteolysis of GST-FldYD-GFP from the FldYD moiety by translocating an internal loop to the protease chamber in an ATP-dependent manner and that, at least, the proteolysis in the C to N direction proceeds processively.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
ATP-dependent proteases, universally conserved in both prokaryotes and eukaryotes, play important roles in diverse cellular processes (for review, Schmidt et al. 1999; Wickner et al. 1999; Zwickl et al. 2000; Gottesman 2003). All ATP-dependent proteases belong to the AAA+ (ATPases associated with diverse cellular activities plus) superfamily and share a conserved ATPase domain of 200–250 amino acid residues (Neuwald et al. 1999; Ogura & Wilkinson 2001; Lupas & Martin 2002). AAA+ proteases usually form barrel-shaped oligomers with a narrow central pore. Pore sizes revealed by X-ray crystal structural analysis are ~10 Å in diameter (Bochtler et al. 2000; Guo et al. 2002), which is too narrow to allow the passage of natively folded proteins. Translocation and engulfment of the substrate proteins is necessary for proteolysis, since the active sites are located in a chamber within the barrel (Bochtler et al. 1997; Groll et al. 1997; Wang et al. 1997). Structural and biochemical studies have suggested that substrate proteins are recognized near the central pore and unfolded at its entrance before being threaded through the apical pore into the protease chamber (Ortega et al. 2000; Reid et al. 2001; Zhang et al. 2002; Matouschek 2003; Sauer et al. 2004). The precise molecular mechanisms of substrate unfolding and threading are largely unknown.

In Escherichia coli, there are four cytosolic (ClpAP, ClpXP, HslUV and Lon) and one membrane-bound (FtsH) ATP-dependent proteases (Gottesman 2003). FtsH is responsible for quality control of membrane proteins by degrading misassembled membrane proteins. It also contributes to the control of cellular functions through degradation of a subset of cytoplasmic regulatory proteins (for review, Gottesman 2003; Akiyama et al. 2004). FtsH contains two transmembrane segments at its N-terminus, which are essential for homo-oligomerization in addition to anchoring to the membrane (Akiyama & Ito 2001; Ito & Akiyama 2005). The large C-terminal cytosolic region consists of the ATPase and protease domains. Crystal structures of the AAA ATPase domains of E. coli and Thermus thermophilus FtsHs have been solved (Krzywda et al. 2002; Niwa et al. 2002). An electron microscopic study indicated that FtsH forms ring-shaped homo-oligomers similar to other AAA+ proteases (Shotland et al. 1997).

In general, AAA+ proteases initiate proteolysis from a degradation signal located at either of the substrate polypeptide's terminal regions and degradation proceeds processively (Lee et al. 2001; Reid et al. 2001; Chiba et al. 2002). It has been shown that ClpAP and ClpXP degrade model proteins with tags located in the interior of the primary sequence (Hoskins et al. 2002). The 26S proteasome requires an unstructured region within a substrate polypeptide, in addition to a polyubiquitin tag, for efficient degradation, and the unstructured region can be located at an internal site (Prakash et al. 2004). Although direct evidence has not been obtained, these results suggest that ATP-dependent proteases are able to initiate proteolysis from internal sites of substrate polypeptides. Recently, Liu et al. (2003) showed that natively disordered substrates lacking a free terminus have access to the proteolytic chamber of 20S and 26S proteasomes for endoproteolysis. However, this endoproteolysis of circular polypeptides is not processive nor is it ATP-dependent. Therefore, it is not clear whether or not AAA+ proteases are able to initiate processive degradation from internal sites of a polypeptide.

During the attempt to develop a convenient system for monitoring substrate unfolding and degradation by using E. coli flavodoxin (Fld) containing a non-covalently bound fluorescence compound, flavin mononucleotide (FMN), we have found that FtsH can degrade apo-Fld, but not the more stable holo-Fld. We constructed a mutant Fld, carrying a substitution of Tyr94 to Asp (FldYD), which has a lower affinity for FMN presumably due to the loss of the interactions between Tyr94 and FMN. Interestingly, FldYD itself, in which no additional recognition tag such as SsrA was attached, was efficiently degraded by FtsH. To reveal the directionality of FldYD degradation by FtsH, we have constructed several fusion proteins of FldYD with either glutathione S-transferase (GST) or green fluorescent protein (GFP), or both. The experiments described here show that free termini of FldYD do not directly contribute to substrate recognition by FtsH. The results also indicate that FtsH can initiate proteolysis from internal sites within the FldYD moiety of the fusion proteins and that the proteolysis proceeds processively, at least, in the C to N direction.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
E. coli apo-Fld as a model substrate for FtsH protease

We purified wild-type E. coli Fld, prepared holo-Fld and apo-Fld, and examined them for degradation by FtsH. Results showed that apo-Fld was efficiently degraded by FtsH in the presence of ATP, while holo-Fld was not significantly degraded under the same conditions (T. Okuno, K. Yamanaka and T. Ogura, unpublished observation). Preparation of apo-Fld includes denaturation of purified Fld, dialysis to remove FMN, and renaturation. To obtain an apo-form of Fld more conveniently, we constructed a mutant Fld in which Tyr94 was mutated to Asp (designated FldYD hereafter). It has been shown that the Y94D mutation decreases dramatically the affinity of Fld for FMN in the case of the Anabaena protein (Lostao et al. 2000). Indeed, less than 5% of the purified FldYD was found to contain FMN. However, the circular dichroism spectral analysis suggested that the secondary structure is not perturbed by the mutation. FldYD was efficiently degraded by FtsH (T. Okuno, K. Yamanaka and T. Ogura, unpublished observation).

FtsH degrades FldYD fusion protein lacking the free N-terminus of FldYD

As described above, FtsH degraded apo-forms of Fld. How does FtsH recognize apo-Fld and initiate its degradation? Previously, we constructed several fusion proteins of physiological substrates, to which GST or GFP, or both were attached. Using these engineered substrates, we were able to analyze substrate recognition and the directionality of proteolysis by FtsH (Okuno et al. 2004). Thus, it is useful to construct Fld fusion proteins with GST and GFP to reveal the directionality of substrate degradation by FtsH.

Accordingly, we constructed several FldYD fusion proteins (Fig. 1). First, we examined a GST-FldYD fusion protein. GST-FldYD was incubated with FtsH at 27 °C. As shown in Fig. 2A, FtsH degraded GST-FldYD efficiently in the presence of ATP but not in the presence of ADP. FtsH does not recognize GST as a substrate itself (Okuno et al. 2004). This indicates that FtsH does not require the free N-terminus of FldYD for degradation.


Figure 1
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  		Figure 1  Construction of FldYD fusion proteins. Fusion proteins used in this work are shown schematically.

 

Figure 2
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  		Figure 2  Degradation of GST-FldYD and its fragments by FtsH. GST-FldYD (2.3 µM) was incubated with FtsH (0.23 µM) in the presence of 3 mM ATP at 27 °C. (A) CBB staining. (B) Western blotting with anti-GST antibodies. (C) Remaining fusion protein estimated by SDS-PAGE analysis and GST activity assay. Amounts of the intact substrate remaining in the reaction mixtures in the presence of ATP (closed triangles) or ADP (open triangles) were quantitated by densitometry. GST activity of the reaction mixtures in the presence of ATP (closed circles) or ADP (open circles) was measured as described in Experimental procedures. (D) GST-containing fragments produced by degradation of GST-FldYD (~0.2 µM) were incubated with FtsH (0.23 µM) in the presence of 5 mM ATP at 27 °C, and analyzed by SDS-PAGE followed by Western blotting with anti-GST antibodies.

 
When GST-FldYD was incubated with FtsH, discrete proteolytic fragments appeared and accumulated in a time-dependent manner. Western blotting analysis indicated that all these fragments contained the GST domain (Fig. 2B). The sizes of these fragments were estimated to be approximately 32.9, 32.0 30.3, 28.9 and 27.3 kDa. No fragment smaller than GST itself was observed by Western blotting analysis and staining with Coomassie Brilliant Blue (CBB). We also measured GST activity at indicated time points during the reaction. After a 100-min incubation, > 90% of the full length GST- FldYD disappeared, but ~30% of the GST activity still remained (Fig. 2C). These results indicated that ~70% of GST-FldYD was degraded into the GST portion of the fusion protein.

Fragments accumulated in these reactions could be purified using a glutathione-Sepharose column. It was found that all of the fragments bound to the column (Fig. 2D, time 0), indicating that they have a natively folded active GST domain. The purified fragments were examined for further degradation by FtsH. Results showed that they were resistant to further proteolysis by FtsH (Fig. 2D). These results indicate that FtsH degrades GST-FldYD in the C to N direction. It is also indicated that the degradation is in part aborted to produce the fragments containing the whole GST domain, and that these fragments are no longer recognized and degraded by FtsH. Therefore, the complete digestion of the majority of GST-FldYD results from the processive proteolysis in the C to N direction.

A free FldYD C-terminus is not required for degradation by FtsH

Next, we prepared a FldYD-GFP fusion protein, in which the C-terminus of FldYD is blocked by GFP (Fig. 1), from GST-FldYD-GFP by PreScission protease cleavage. FtsH is unable to degrade the GFP moiety in fusion proteins (Herman et al. 2003; Okuno et al. 2004). Degradation of FldYD-GFP by FtsH was analyzed by Western blotting using anti-GFP antibodies. As shown in Fig. 3A, FldYD-GFP was degraded by FtsH in the presence of ATP, but not in the presence of ADP, with several discrete fragments detected by anti-GFP antibodies. The sizes of these fragments were estimated to be approximately 43.4, 41.5, 39.1, 36.9, 36.1, 33.4, 31.4 and 30.3 kDa, respectively.


Figure 3
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  		Figure 3  Degradation of FldYD-GFP by FtsH. FldYD-GFP (2.3 µM) was incubated with FtsH (0.23 µM) in the presence of 3 mM ATP at 27 °C. (A) Samples were analyzed by SDS-PAGE followed by Western blotting with anti-GFP antibodies. (B) GFP fluorescence change during the degradation reaction.

 
The ~39.1 kDa and larger fragments appeared to be decreased during the incubation, suggesting that these fragments were further degraded by either resumption of processive proteolysis from transient posing or re-initiation of proteolysis by another FtsH after being released abortively.

No decrease in the intensity of GFP fluorescence was observed during the reaction (Fig. 3B), indicating that proteolysis is limited to the FldYD moiety of the fusion protein. Therefore we conclude that FtsH degrades the FldYD moiety of FldYD-GFP and leaves the GFP moiety undigested. Since FtsH degraded GST-FldYD in the C to N direction, and degraded the N-terminal FldYD domain of FldYD-GFP, it is likely that FtsH can catalyze degradation of the FldYD fusion proteins in either direction.

FtsH does not require a free FldYD terminus to initiate degradation, and proteolysis can be initiated from internal sites

The proteolysis results for GST-FldYD and FldYD-GFP raise the possibility that FtsH may be able to initiate proteolysis from either terminus of FldYD in these fusion proteins. Alternatively, FtsH may not require a free terminus in its substrate polypeptides and may be able to initiate proteolysis from an internal site(s) in these substrates.

To test the importance of free termini in substrate polypeptides for the initiation of the proteolytic reaction of FtsH, we examined degradation of a GST-FldYD-GFP fusion protein, in which the FldYD domain is sandwiched by GST and GFP. It was found that GST-FldYD-GFP was degraded by FtsH in the presence of ATP but not in the presence of ADP (Fig. 4A,B). The preparation was contaminated with some in vivo proteolytic fragments containing the complete GST domain due to low expression of this fusion protein. Fragments detected by anti-GST (Fig. 4A) or anti-GFP (Fig. 4B) antibodies accumulated in a time-dependent manner, and their sizes were identical to those of the fragments observed when GST-FldYD and FldYD-GFP were degraded by FtsH, respectively (compare with Figs 2B and 3A).


Figure 4
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  		Figure 4  Degradation of GST-FldYD-GFP by FtsH. Degradation was analyzed by SDS-PAGE followed by Western blotting with anti-GST (A) and anti-GFP (B) antibodies. Fusion protein (2.3 µM) was incubated with FtsH (0.23 µM) in the presence of 3 mM ATP at 27 °C. (C) GFP fluorescence change during the degradation reaction.

 
The GFP fluorescence did not change during the degradation reaction (Fig. 4C), indicating that the GFP domain of the fusion protein was totally undigested. This clearly excludes the possibility that the fragments containing the GST domain detected in Fig. 4A were produced by processive proteolysis from the C-terminus of the fusion protein. Taken together, it is evident that FtsH initiates proteolysis from the internal sites of FldYD and that the proteolysis proceeds processively, at least, in the C to N direction.

Requirement of active translocation for degradation of GST-FldYD

Recently, Ondrovicováet al. (2005) reported that mitochondrial Lon protease initiates proteolysis at internal sites of folded substrate proteins first, and then subsequent degradation proceeds processively along the primary polypeptide sequence. These results are similar to our present results with GST-FldYD-GFP described above. Ondrovicováet al. (2005) have proposed a ring-opening mechanism, which permits direct access of a folded substrate to the active site of Lon protease. If the initial cleavage occured at internal sites of folded substrates by the ring-opening mechanism, translocation of unfolded substrates would not be required. For translocation of polypeptides in FtsH, we showed that ATP hydrolysis is required, and that the conserved pore residues of the ATPase ring are essential (Yamada-Inagawa et al. 2003).

We examined the requirement of ATP hydrolysis for degradation of GST-FldYD. As shown in Fig. 5, ATP{gamma}S did not support the degradation. It should be pointed out that no sign of a single cleavage was observed in the presence of ATP{gamma}S. We also examined a pore mutant FtsH carrying a substitution of Phe228 to Ala (FtsHFA) for its ability to degrade GST-FldYD, and observed no cleavage of the fusion protein (Fig. 5). These results indicate that proteolysis of FldYD does require ATP hydrolysis and translocation of polypeptides. The pore mutant FtsH's inability to cleave GST-FldYD strongly argues against the ring-opening mechanism for the initial cleavage.


Figure 5
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  		Figure 5  ATP hydrolysis- and translocation-dependent degradation of GST-Fld.YD GST-FldYD (2.3 µM) was incubated with FtsH (0.23 µM) in the presence of 3 mM ATP or ATP{gamma}S, or with FtsHFA (0.23 µM) in the presence of 3 mM ATP at 27 °C. Samples were analyzed by SDS-PAGE followed by Western blotting with anti-GST antibodies.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
It has been suggested that FtsH recognizes tags at either terminus of its membrane protein substrates and initiates degradation from the terminus (Chiba et al. 2002). We previously noted that the degradation of {lambda}CII, a physiological substrate of FtsH containing the recognition signal at the C-terminus (Kobiler et al. 2002), was completely inhibited by fusing GFP to the C-terminus, but not by fusing GST to the N-terminus (Okuno et al. 2004). Results with another substrate, {sigma}32, were different from those with {lambda}CII. The fusion of GST to the N-terminus of {sigma}32 inhibited degradation, but the fusion of GFP to the C-terminus did not inhibit degradation of the {sigma}32 moiety of the fusion protein, suggesting that the degradation of {sigma}32 proceeds in the N to C direction.

FtsH degraded GST-FldYD, FldYD-GFP and even GST-FldYD-GFP. The fragments produced by the GST-FldYD-GFP degradation (Fig. 4) are identical to the sum of those produced by the degradation of GST-FldYD (Fig. 2) and FldYD-GFP (Fig. 3). These results indicate that the manner of degradation of GST-FldYD-GFP is essentially the same as those of GST-FldYD and FldYD-GFP. Therefore, we conclude that FtsH can initiate proteolysis from the internal FldYD domain in the presence of ATP. The key reaction in this type of proteolysis from internal sites must be the first endolytic cleavage of the substrate polypeptides. It requires ATP hydrolysis, since ATP{gamma}S does not support the reaction (Fig. 5). This provides firm evidence for a conceptually new ATP-dependent proteolysis from internal sites of substrate proteins.

To achieve endolytic cleavage at internal sites, there are two possibilities. One possibility is that an internal loop of the substrate polypeptides is translocated into the proteolytic chamber through the central pore of the FtsH ring. Another possibility is that the FtsH ring opens and directly cleaves the folded substrate. Ondrovicováet al. (2005) have proposed the ring-opening mechanism for mitochondrial Lon protease. Although this type of mechanism is attractive, it is unlikely for FtsH. Instead, the pore mutant FtsH, which is defective in translocation of polypeptides, did not produce endolytically cleaved fragments in support of the former possibility. Therefore, it is most likely that active translocation of an internal loop to the proteolytic chamber is necessary for the first endolytic cleavage, which precedes subsequent proteolysis (Fig. 6). The initial cleavage at internal sites of substrates by Lon might also occur by translocation of an internal loop into the protease chamber. Although it is likely that Lon recognizes folded substrates, there is no direct evidence for the folding state of substrates at the moment of initial cleavage.


Figure 6
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  		Figure 6  Proposed model for the initial stage of proteolysis of GST-FldYD-GFP by FtsH. The FldYD moiety of the GST-FldYD-GFP fusion protein is recognized by FtsH. The FldYD domain is unfolded and an internal loop (red) is translocated into the protease chamber of FtsH through the pore of the ATPase ring, leading initiation of proteolysis from an internal site of the fusion protein.

 
Recent studies have revealed that some ATP-dependent proteases allow passage of a pair of polypeptide chains simultaneously. It was shown that a disulfide-bonded pair in a model substrate can be passed through the axial pore of ClpXP into the protease chamber (Burton et al. 2001), and that the pore of the proteasome also allows the concurrent passage of at least three stretches of polypeptide chain (Lee et al. 2002). Since the pore size of the ATPase domain of FtsH deduced from the crystal structure and modeling is ~10 Å in diameter (Krzywda et al. 2002; Niwa et al. 2002), dynamic structural changes of the pore must be needed to pass an internal loop of a polypeptide through the pore.

Recently, Liu et al. (2003) showed that both the 20S and the 26S proteasomes catalyze endoproteolytic cleavage at internal sites of circular polypeptides containing natively disordered domains. However, this reaction, which includes the translocation of a loop of circular polypeptides into the protease chamber, does not require ATP hydrolysis, as is evident from the fact that the 20S proteasome catalyzes it. Hoskins et al. (2002) indicated that ClpAP and ClpXP degrade substrates with recognition tags located in the interior of the primary sequence. Their results do not demonstrate the location of the initiation site for proteolysis or its direction. Prakash et al. (2004) found that the 26S proteasome requires an unstructured region, in addition to the polyubiquitin recognition tag, for efficient degradation. This type of unstructured region can be placed at a position remote from the polyubiquitin tag. Although they further showed that an unstructured region placed at the C-terminus of the substrate serves as the proteolytic initiation site, the initiation site and the direction of proteolysis of a polypeptide containing an internal unstructured region has not been experimentally confirmed. Here we show clearly that FtsH initiates degradation from internal sites of the FldYD moiety in fusion proteins. This indicates that a free terminus in the substrate proteins is not always required for ATP-dependent proteolysis by AAA+ proteases.

Results suggest that the degradation of GST-FldYD proceeds processively in the C to N direction after the endolytic cleavage. A single FtsH oligomer may cleave the FldYD fusion protein at an internal site, and then catalyze subsequent processive degradation without releasing the cleaved polypeptides. Alternatively, the fragments produced by endoproteolytic cleavage by an FtsH oligomer may be recognized and degraded by other FtsH oligomers. Although FtsH degraded most fractions of the GST-FldYD completely, ~30% of GST-FldYD was partially degraded and fragments containing the complete GST domain remained undigested (Fig. 2). These fragments were no longer digested by FtsH. It is possible that FtsH at some frequency fails to initiate unfolding of the GST domain and releases fragments containing the GST domain. It has been demonstrated that similar partial degradation occurs in general, when AAA+ proteases encounter a stable structural element (Sauer et al. 2004).

It is still unclear, however, whether or not the degradation in the N to C direction proceeds processively. Degradation of FldYD-GFP by FtsH is always incomplete, producing several discrete sizes of fragments, and FtsH did not degrade the tightly folded GFP moiety (Fig. 6). Since the ~39.1 kDa and larger fragments were further degraded by FtsH, it is suggestive that the degradation of FldYD-GFP in the N to C direction also proceeds processively. Further careful experiments are needed to elucidate the possibility that the same FtsH oligomer can mediate bi-directional processive proteolysis of the FldYD fusion protein.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains and plasmids

DH5{alpha} (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoR{Delta}(argF-lac)U169{phi}80lacZ{Delta}M15) and AR5771 (BL21[DE3]sfhC21{Delta}ftsH3::kan{Delta}hflKC3::tet) were used as host strains for construction of plasmids and for expression of genes cloned on plasmids. Cells were grown in L medium (10 g of tryptone, 5 g of yeast extract and 5 g of NaCl/L, pH 7.4). Ampicillin (100 µg/mL) was added for growing strains carrying plasmid.

To construct the fusion gene coding for GST-Fld, the fldA gene was amplified by PCR using a purified chromosome DNA as a template and inserted into the BamHI-XhoI site of the multicloning site of pGEX-6p-1 (Amersham Bioscience) that is located downstream of the glutathione S-transferase gene. To construct the fusion gene coding for GST-Fld-GFP, the fldA gene lacking a stop codon was inserted into the EcoRI-SalI site of pGEX-6p-1 and then the gfp gene was inserted into the SalI-EagI site. There is a linker sequence corresponding to six amino acid (EFGRLE) sequence between the fldA gene and the gfp gene. A mutation (Tyr94 to Asp) was introduced in the fldA gene using mutagenic oligonucleotides (5'-CGGCCGCTCGAGTTACGCTTCAATGGCAGCACGCAATTTACACATCGCGTTCTTTTCCAG-3' and 5'-GAGAGGGGATCCATGACTGACAAAATG-3').

Protein preparation

Wild-type and mutant FtsHs were expressed in E. coli strain AR5771 and purified by the procedures previously described (Yamada-Inagawa et al. 2003). Purified FtsH samples were stored at –80 °C. GST fusion proteins were expressed in DH5{alpha}. Cells were grown at 30 °C, and expression of GST-FldYD and GST-FldYD-GFP was induced by the addition of IPTG (1 mM) at 50 Klett units. After 3 h, cells were harvested by centrifugation at 4 °C, lyzed by sonication, and centrifuged for 20 min at 10 000 r.p.m. The supernatant was loaded on a HiTrapQ column (Amersham Bioscience) in buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol) and eluted with a linear gradient from 150 mM to 1 M NaCl. Fractions containing GST fusion proteins were loaded on a GSTrap column (Amersham Bioscience) and eluted with buffer A containing 20 mM reduced glutathione. Pooled fractions were loaded on a Superdex 200 HR 10/30 column (Amersham Bioscience). The GST moiety of these GST fusion proteins was cleaved off by PreScission protease (Amersham Bioscience) to produce FldYD and FldYD-GFP. These proteins contain an additional five amino acid (GPLGS) sequence at the N-terminus that is not present in authentic Fld. The reaction mixture was loaded on a Superdex 200 column in buffer A. Purified proteins were stored at –80 °C.

To prepare GST-containing fragments produced by degradation of GST-FldYD, GST-FldYD (0.012 µM) was digested with FtsH (0.14 µM) in the presence of 10 mM ATP at 27 °C for 2 h. A 1.2-mL reaction mixture, which contained no detectable full length GST-FldYD, was loaded on a GSTrap column and eluted with buffer A containing 20 mM reduced glutathione.

Protein degradation assays

Purified FldYD fusion proteins were incubated with FtsH at 27 °C in reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM Mg(OAc)2, 25 µM Zn(OAc)2,1 mM DTT, 0.1% NP-40) plus 3 mM ATP, ADP, or ATP{gamma}S. At the indicated time points, an aliquot was removed from the reaction mixture, and the reaction was terminated by the addition of the sample buffer of electrophoresis. Samples were analyzed by SDS-PAGE followed by staining with CBB or immunoblotting using anti-GST (HRP conjugated; Amersham Bioscience) or anti-GFP (Roche) antibodies and detection by ECL. Amounts of substrate were quantitated by densitometry.

GST activity assay

GST fusion proteins (2.3 µM) were incubated with FtsH (0.23 µM) at 27 °C in the reaction buffer with 3 mM ATP or ADP. At the indicated time points, samples (20 µL) were removed and the reaction was stopped by the addition of 5 µL of 0.5 M EDTA (pH 7.5) on dry ice. GST activity was assayed in 750 µL of the GST reaction buffer (100 mM sodium phosphate pH 6.5, 1 mM reduced glutathione, 1 mM 1-chloro-2,4-dinitrobenzene) by measuring a rate of increase in A340. Amounts of active GST were quantitated from a calibration curve.

Spectrometry

Fluorescence of GFP (emission at 510 nm; excitation at 488 nm) was measured by a spectrofluorometer (Jasco FP-750).


   Acknowledgements
 
We thank Satomi Fukunaga for excellent technical assistance, Chiyome Ichinose and Yuki Kawata for secretarial assistance. We also thank Anthony J. Wilkinson for critical reading of the manuscript and stimulating discussion. This work was supported, in part, by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by the Core Research for Evolutional Science and Technology (CREST) program grant from the Japan Science and Technology Agency (JST).


   Footnotes
 
Communicated by: Hiroji Aiba

Present address: aDepartment of Physical Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan. Back

* Correspondence: E-mail: ogura{at}gpo.kumamoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Akiyama, Y. & Ito, K. (2001) Roles of homooligomerization and membrane association in ATPase and proteolytic activities of FtsH in vitro. Biochemistry 40, 7687–7693.[Medline]

Akiyama, Y., Ito, K. & Ogura, T. (2004) FtsH protease. In: Handbook of Proteolytic Enzymes, 2nd edn (eds A. J. Barrett, N. D. Rawlings & J. F. Woessner), pp. 794–798. Amsterdam: Elsevier Academic Press.

Bochtler, M., Ditzel, L., Groll, M. & Huber, R. (1997) Crystal structure of heat shock locus V (HslV) from Escherichia coli. Proc. Natl. Acad. Sci. USA 94, 6070–6074.[Abstract/Free Full Text]

Bochtler, M., Hartmann, C., Song, H.K., Bourenkov, G.P., Bartunik, H.D. & Huber, R. (2000) The structures of HslU and the ATP-dependent protease HslU-HslV. Nature 403, 800–805.[CrossRef][Medline]

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Received: 13 November 2005
Accepted: 12 December 2005




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