New insights into the ATP-dependent Clp protease: Escherichia coli and beyond.

Proteolysis functions as a precise regulatory mechanism for a broad spectrum of cellular processes. Such control impacts not only on the stability of key metabolic enzymes but also on the effective removal of terminally damaged polypeptides. Much of this directed protein turnover is performed by proteases that require ATP and, of those in bacteria, the Clp protease from Escherichia coli is one of the best characterized to date. The Clp holoenzyme consists of two adjacent heptameric rings of the proteolytic subunit known as ClpP, which are flanked by a hexameric ring of a regulatory subunit from the Clp/Hsp100 chaperone family at one or both ends. The recently resolved three-dimensional structure of the E. coli ClpP protein has provided new insights into its interaction with the regulatory/chaperone subunits. In addition, an increasing number of studies over the last few years have recognized the added complexity and functional importance of ClpP proteins in other eubacteria and, in particular, in photosynthetic organisms ranging from cyanobacteria to higher plants. The goal of this review is to summarize these recent findings and to highlight those areas that remain unresolved.

The ATP-dependent Clp protease is essential for acclimation to UV-B and low temperature in the cyanobacterium Synechococcus.

ClpP is the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. Cyanobacterial ClpP protein is encoded by a multigene family, producing up to four distinct isozymes. We have examined the importance of the first ClpP protein (ClpP1) isolated from the cyanobacterium Synechococcus sp. PCC 7942 for acclimation to ecologically relevant UV-B and low-temperature regimens. When the growth light of 50 mumol photons m-2 s-1 was supplemented with 0.5 W m-2 UV-B for 8 h, the constitutive level of ClpP1 rose eightfold after an initial lag of 1 h. Wild-type cells readily acclimated to this UV-B level, recovering after the initial stress to almost the same growth rate as that before UV-B exposure. Growth of a clpP1 null mutant (delta clpP1), however, was severely inhibited by UV-B, being eight times slower than the wild type after 8 h. In comparison, ClpP1 content increased 15-fold in wild-type cultures shifted from 37 degree C to 25 degree C for 24 h. Wild-type cultures readily acclimated to 25 degree C after 24 h, whereas the delta clpP1 strain did not and eventually lost viability with prolonged cold treatment. During acclimation to either UV-B or cold, photosynthesis in the wild type was initially inhibited upon the shift but then recovered. Photosynthesis in delta clpP1 cultures, however, was more severely inhibited by the stress treatment and failed to recover. Acclimation was also monitored by examining the exchange of photosystem II reaction centre D1 proteins that occurs in wild-type Synechococcus during conditions of excitation stress. During both cold and UV-B shifts, wild-type cultures replaced the acclimative form of D1 (D1:1) with the alternative D1 form 2 (D1:2) within the first hours. Once acclimated to either 25 degree C or 0.5 W m-2 UV-B, D1:2 was exchanged back for D1:1. In delta clpP1 cultures, this second exchange between D1 forms did not occur, with D1:2 remaining the predominant D1 form. Our results demonstrate that the ATP-dependent Clp protease is an essential component of the cold and UV-B acclimation processes of Synechococcus.

Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation.

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1). A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.