Biosci. Biotechnol. Biochem., 70 (2), 325–331, 2006
Review
A Polyphosphate-Lon Protease Complex in the Adaptation of Escherichia coli to Amino Acid Starvation Akio KURODA1;2 1
Department of Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan 2 PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Cells must balance energy-efficient growth with the ability to adapt rapidly to sudden changes in their environment. For example, in an environment rich in amino acids, cells do not expend energy for making amino acid biosynthetic enzymes. However, if the environment becomes depleted of amino acids (nutritional downshift), cells will be exposed to a lack of both the amino acid biosynthetic enzymes and the amino acids required to make these enzymes. To solve this dilemma, cells must use their own proteins as sources of amino acids in response to the nutritional downshift. Once amino acid biosynthetic enzymes start to accumulate, the cell is able to produce its own amino acids, and a new growth phase begins. In Escherichia coli, amino acid starvation leads to the accumulation of an unusual molecule, polyphosphate (polyP), a linear polymer of many hundreds of orthophosphate residues. Protein degradation in this bacterium appears to be triggered by the accumulation of polyP. PolyP forms a complex with the ATP-dependent Lon protease. The formation of a complex then enables Lon to degrade free ribosomal proteins. Certain very abundant ribosomal proteins can be the sacrificial substrates targeted for degradation at the onset of the downshift. Here I propose to call the polyP-Lon complex the ‘‘stringent protease,’’ and I discuss new insights of protein degradation control in bacteria. Key words:
stringent response; polyphosphate; amino acid starvation; Escherichia coli; Lon protease
I. Increase in Protein Turnover during Amino Acid Starvation By the 1950s, it was known that the rate of protein degradation increases under conditions of amino acid starvation in prokaryotes such as the bacterium Escherichia coli.1,2) It was also known that amino acid starvation causes coupled cessation of protein synthesis and stable RNA synthesis, a process called the ‘‘stringent response.’’3) These metabolic responses are very
important to allow cells to escape from amino acid starvation and to adapt to new environmental conditions. If the environment is suddenly depleted of amino acids, new enzymes can be synthesized only from amino acids liberated by protein turnover.4) In fact, the Salmonella typhimurium peptidase mutant can not adapt to such changes in its environment.4) Thus, during periods of starvation, there is increased degradation of otherwise stable proteins. Starvation-induced proteolysis is an energy-dependent process requiring the hydrolysis of adenosine triphosphate (ATP).5,6) In yeast and animal cells, the bulk degradation of proteins in response to starvation and cellular differentiation occurs by a ubiquitin-style conjugation system.7–9) In bacteria, however, the proteases and regulatory factors responsible for protein degradation during starvation have not been identified, and little is known about which proteins are degraded.5,6)
II. The Stringent Response Leads to Accumulation of Inorganic Polyphosphate Inorganic polyphosphate (polyP) is a linear polymer of many hundreds of orthophosphate (Pi ) residues linked together by ATP-like high-energy phosphoanhydride bonds.10) In 1995, my colleagues and I in Arthur Kornberg’s laboratory found that the level of polyP in E. coli is very low during the exponential phase, but that the concentration increases up to 1,000-fold in response to amino acid starvation.11) The stringent response depends on guanosine tetraphosphate (ppGpp), a key signaling molecule in starved or nutritionally stressed cells.12) Synthesis of ppGpp is induced in the presence of idling ribosomes and uncharged transfer RNAs (tRNAs without their attached amino acids).12) This molecule integrates many of the transcriptional effects caused by amino acid starvation. It shuts down the transcription of genes encoding ribosomal proteins and proteins involved in rapid growth, and it switches on genes required for biosynthetic pathways that replenish depleted metabolites.12) Intriguingly, ppGpp is also required for the accumulation of polyP, because an E. coli relA spoT mutant that
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Amino acid starvation
Ribosome Amino acids Ribosomal proteins
Stringent response
degradation Lon
pppGpp
PP P
Inhibition PP P
PP P PolyP
PPX Pi ATP PPK
PolyP-Lon complex (stringent protease) Fig. 1. The PolyP-Related Stringent Network for Protein Degradation. During amino acid starvation in E. coli, there is increased production of (p)ppGpp along with the ‘‘stringent response.’’ The (p)ppGpp inhibit the activity of PPX. In E. coli, the level of polyP is very low in the exponential phase, but it increases up to 1,000-fold in response to amino acid starvation. PolyP forms a complex with the ATP-dependent Lon protease, and this complex degrades free ribosomal proteins. Amino acids are then made available to the cell by cytoplasmic peptidases that cleave the short peptides released by Lon. The cell is then able to adjust to nutrient-poor conditions by de novo production of amino acid biosynthetic enzymes from the released amino acids. The oligomeric structure of the polyP-Lon complex is predicted based on the molecular weight obtained from dynamic light-scattering measurements.
fails to produce pppGpp and ppGpp is deficient in the accumulation of polyP.11) PolyP kinase (PPK, EC 2.7.4.1) reversibly transfers the terminal Pi group of ATP to short-chain polyP, generating long-chain polyP, and exopolyphosphatase (PPX, EC 3.6.1.11) degrades polyP into Pi .10) Increasing the amounts of pppGpp and ppGpp has no influence on the activity of PPK, but these compounds strongly inhibit PPX. For example, at 100 mM, pppGpp inhibits PPX by 90%; ppGpp also inhibits PPX, but less strongly.11) PPX shares sequence homology with guanosine pentaphosphate hydrolase (GppA, EC 3.6.1.40), but PPX only weakly degrades pppGpp, and it does not appear to have a significant role as a guanosine pentaphosphate hydrolase in vivo. Thus pppGpp competitively inhibits PPX activity (Ki value of 10 mM).11) Therefore, the current model for polyP accumulation during the stringent response is that PPK and PPX activities are constitutively expressed, but when pppGpp and ppGpp builds up in the cells during amino acid starvation, polyP accumulates due to inhibition of PPX activity (Fig. 1). This accumulation of polyP during amino acid starvation is reversed if growth is allowed to resume. Harold reported that polyP is rapidly degraded and that the resulting Pi is quantitatively transferred to the nucleic acid fraction, suggesting that cessation of nucleic acid synthesis and continued assimilation of Pi from the medium results in the accumulation of polyP.13) According to his model, inhibitors of nucleic acid
synthesis should stimulate polyP accumulation. In fact, novobiocin (a specific inhibitor of DNA gyrase) and rifampicin (an inhibitor of RNA polymerase) stimulate polyP accumulation, while inhibitors of protein synthesis or cell wall synthesis do not.14) Recently studies with a ppx mutant showed that it has a significantly decreased rate of polyP degradation, but the level of polyP is not as high as expected. Finally, during amino acid starvation, RNA synthesis is inhibited by (p)ppGpp.12) These findings suggest that the inhibition of nucleic acid synthesis by stringent factors plays an important role in polyP accumulation.
III. PolyP Is Necessary for Adaptation to a Nutritional Downshift (amino acid starvation) An E. coli mutant deficient in PPK, the principal enzyme for the synthesis of polyP, shows an extended lag in growth when shifted from a nutrient-rich to a nutrient-poor medium (nutritional downshift) (Fig. 2).15) The addition of amino acids to a nutrient-poor medium abolishes this growth lag. The wild type transiently accumulated polyP at the onset of growth on nutrientpoor medium, whereas the ppk mutant did not (Fig. 2).15) To determine whether the ppk mutant affects the expression of amino acid biosynthetic genes, we constructed a hisG-lacZ transcriptional fusion. Within 5 h, the wild type induced hisG-lacZ in response to the
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downshift, but this was not observed in the mutant. Northern blot analysis, however, revealed that the mutant still transcribes the hisG-lacZ gene at 20% of the wild-type level 3 h after the downshift. The addition of low concentrations (1.25 mg/l) of amino acids to the nutrient-poor medium rescued induction of hisG-lacZ expression in the mutant.15) Because amino acids are required for the production of enzymes mediating adaptation to the downshift, protein degradation is stimulated during amino acid starvation or nutritional
Fig. 2. Cell Growth and PolyP Accumulation during the Nutritional Downshift. E. coli MG1655 (wild type) and CF5802 (ppk ppx double mutant) cells were subjected to a nutritional downshift from 2YT rich medium to Mops minimal medium (nutrient-poor). PolyP accumulated in response to the downshift in the wild type (open circles) but not in the mutant (open squares). Growth was measured as the optical density at 600 nm (OD600 ) in the wild type (filled circles) and the ppk ppx double mutant (filled squares).
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downshift. Indeed, the wild type increased protein turnover in response to the nutritional downshift, but it did not increase in the mutant (Fig. 3). The ppk mutant appeared to fail in the activation of a pathway (proposed below) that supplies amino acids when the environment suddenly becomes depleted of them.
IV. PolyP-Lon Complex: A Missing Link between the Stringent Response and Increased Protein Turnover In E. coli, more than 90% of cytoplasmic protein degradation is energy-dependent.5) The ATP-dependent protease Lon, conserved from bacteria to humans, is responsible for the rapid turnover of both abnormal and naturally unstable proteins.16,17) Lon and two other proteases (ClpAP and ClpXP) are responsible for 70% to 80% of the energy-dependent protein degradation in E. coli.5) We found that mutations in both the Lon and Clp proteases produce the same phenotype as ppk mutations and that the addition of amino acids overcomes the block in these cases.18) Because the concentration of ATP after the downshift was similar in the wild type and the ppk mutant, polyP is not likely to serve as an energy source. The rate of protein turnover for the lon clp ppk triple mutant was nearly identical to that of the lon clp double mutant, which suggests that polyP operates in the same pathway for protein degradation as the Lon and Clp proteases. Moreover, purified Lon binds to 32 P-labeled polyP (700 Pi residues; P700) even in the presence of 10 mM Pi .18) A filter-binding assay showed that Lon binds to polyP with a dissociation constant (Kd ) of approximately 0.5 nM irrespective of the presence of ATP, and that approximately four molecules of Lon bind to one molecule of polyP.18)
Fig. 3. Degradation of Intracellular Protein during the Nutritional Downshift. [14 C]Leucine was incorporated during exponential growth. The wild type (filled circles) and the ppk ppx double mutant (open circles) were grown on 2YT rich medium and then resuspended in 2YT rich medium (A) or Mops minimal medium (B). Trichloroacetic acid (TCA) soluble counts at a given time are expressed as percentage of the total initially incorporated counts.
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V. Ribosomal Proteins: Sacrifice Substrates Degraded by the PolyP-Lon Complex An E. coli lysate was fractionated by chromatography on phosphocellulose or DEAE-cellulose, and each fraction was subjected to proteolysis by Lon in the presence and absence of polyP.18) Among the many proteins tested, a few were degraded only in the presence of polyP. The NH2 -terminal sequences of these proteins corresponded to those of ribosomal proteins S2, L9, and L13.18) PolyP (P700) at 1 mM (as a polymer) was effective for degradation of the S2 and L13 proteins. In response to amino acid starvation, the levels of polyP become even higher than 14 mM and far exceed those required to degrade these proteins efficiently. PolyP with a shorter chain length (65 Pi residues) was less potent at stimulating Lon-dependent degradation of S2 and other ribosomal proteins, and a 15-residue polyP was inactive. In the presence of polyP, Lon degrades most LiClextracted ribosomal proteins. We purified several large subunit ribosomal proteins (L1, L3, L6, L9, L13, L15, L17, L18, and L24) and subjected them to Lon-mediated degradation in the presence and absence of polyP. Lon degraded L1, L3, L6, and L24 in the presence but not in the absence of polyP (Fig. 4). PolyP also stimulated the degradation of purified L9, L13, and L17, but not L15 or L18. Lon is known to be responsible for the rapid turnover of regulatory proteins, including SulA, a cell division inhibitor produced in response to DNA damage.19,20) In contrast to the ribosomal proteins tested, the degradation of a maltose-binding protein (MBP)-SulA fusion was slightly inhibited in the presence of 1.4 mM polyP.21) Therefore, polyP does not always stimulate Lon-mediated protein degradation. To monitor the in vivo degradation of the ribosomal protein after the downshift, we expressed a S2-V5epitope fusion in the wild type and the ppk mutant.18) S2-V5 fusion was stable without the downshift but was degraded rapidly after the downshift, although only in the wild type; the S2-V5 fusion was stable in both the lon and ppk mutants even after the downshift. Thus the
Fig. 4. Degradation of Ribosomal Proteins by Lon in the Presence of PolyP. Purified ribosomal proteins were incubated with Lon in the presence and absence of polyP700 (1.4 mM as a polymer) at 37 C for the indicated times.
S2 protein is subject to Lon-dependent degradation in the presence of polyP in vivo as well as in vitro. Biochemical analysis suggested that polyP stimulates Lon to degrade specific proteins. Of the many cellular proteins screened as potential substrates, free ribosomal proteins (not associated with intact ribosomes) were found to be preferentially degraded by the polyP-Lon complex. During exponential growth on a rich medium, E. coli contains significant amounts of disassembled (i.e., free) ribosomal proteins (e.g., 17% of S2 is free), but little disassembled ribosomal protein is found when the cells are grown in minimal medium.22) Such free ribosomal proteins might be subject to Lon-dependent degradation. The polyP-Lon did not degrade intact, native ribosomes. We found that polyP-Lon degrades ribosomes treated with RNase.18) The polyP-Lon complex degraded the ribosomal proteins that are released from ribosomes by RNase treatment, but it remains unknown how the intact ribosomes are degraded in vivo. Based on these findings, it appears that the ppGppdependent increase in protein degradation is due to the accumulation of polyP, which stimulates Lon-mediated degradation of free ribosomal proteins. The ribosome acts as a ‘‘starvation sensor,’’ signaling through ppGpp to the cell and even more ‘‘amino acid reserves’’ liberating amino acids from free ribosomal proteins in response to ppGpp (Fig. 1).
VI. How Are Ribosomal Proteins Made Available for Degradation? Purified S2 binds to polyP with a Kd of approximately 12 nM based on a filter-binding assay, and it does so even in the presence of a 10-fold excess of pUC119 DNA or E. coli total RNA (55% and 30% of control respectively).18) In addition, Lon forms a complex with S2 in the presence of polyP, suggesting that binding of S2 to polyP participates in the formation of a substrateprotease complex. Most ribosomal proteins bind to polyP and are degraded by Lon in the presence of polyP.18) The MBP-SulA protein does not bind to polyP and its degradation by Lon is not stimulated by polyP, indicating that polyP binding is necessary for the polyP stimulation of Lon-mediated proteolysis.21) But not all polyP-binding proteins are degraded by Lon in the presence of polyP. For example, PPK, L15, and L18 bind to polyP, but they are not degraded by Lon in its presence. Therefore, it is likely that polyP binding is necessary but not sufficient for stimulation of Lonmediated proteolysis.21) Is it that polyP first binds to Lon and then the polyPLon complex degrades the ribosomal proteins? Alternatively, is it that polyP binds to the ribosomal proteins and then Lon recognizes the polyP-ribosomal protein complex for degradation? In the latter case, polyP can function much like ubiquitin in eukaryotic cells,9) tagging ribosomal proteins for degradation by proteases. To answer these questions, we examined the effect of
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Fig. 5. A New Insight into PolyP-Related Protein Degradation in E. coli. Lon is localized in the nucleoid in the absence of polyP. In response to a nutrient downshift, there is an increase in the concentration of polyP. As a result, Lon is released from the nucleoid to the cytoplasmic space because it has a higher affinity for polyP than DNA in the nucleoid. This release might allow the polyP-Lon complex to access target proteins in the cytoplasm. The binding of Lon by DNA in the absence of polyP might be to restrict its access to the target proteins.
the order of addition on the cleavage of ribosomal protein L24. We found that Lon efficiently degrades L24 when Lon and polyP are mixed together prior to the addition of L24, whereas there is little degradation of L24 when it is first mixed with polyP before the addition of Lon.21) This result indicates that polyP-Lon complex formation is important for the stimulation of protein degradation and that polyP does not act as a tag for Lonmediated degradation. PolyP functions as an adaptor molecule to stimulate a Lon-substrate complex.
VII. PolyP Competes with DNA for Binding to Lon Lon consists of BAB, ATPase, SSD, and proteolytic domains.23) To identify the site of polyP binding on Lon, we generated deletion mutants of Lon fused to the Cterminus of MBP. We found that the polyP-binding site is within the ATPase domain of Lon between amino acids 320 and 437.21) Although Lon was originally identified as a DNA-binding protein,24,25) the location of the DNA-binding site has not been determined. We found that the DNA-binding site also appears to be in the ATPase domain of Lon.21) Because the ATPase domain contains both DNA- and polyP-binding sites, we suspected that polyP can compete with DNA for binding to Lon. Indeed, the formation of a DNA-Lon complex was completely inhibited in the presence of polyP, suggesting that polyP binds to Lon more strongly than DNA. These results suggest that, although Lon can normally form a complex with DNA, Lon forms a complex with polyP when it accumulates under conditions of amino acid starvation. In fact, during the course of purification, we found that MBP-Lon contains
bound DNA. On the contrary, when MBP-Lon was purified from E. coli cells subjected to amino acid starvation, it contained a significant amount of associated polyP.21) The nucleoid is a multi protein-DNA complex in bacteria. To monitor the localization of Lon, we used a Lon-green-fluorescent protein (GFP) fusion protein. We observed that Lon-GFP is present in the nucleoids when the ppk mutant or the wild type grows on a rich medium. Further, in a phoU mutant, which accumulates polyP to a level several hundred-fold higher than the wild type, Lon-GFP associates with polyP and is present in polyP granules. These results suggest that Lon is normally localized in the nucleoid but is released in response to polyP accumulation (Nomura and Kuroda, unpublished results).
VIII. Perspective: New Insights into PolyPRelated Protein Degradation in E. coli Goff and Goldberg reported that overproduction of Lon causes the proteolysis of normal proteins in cells and is lethal to the host cells.26) We also observed that E. coli growth stops immediately when Lon is overproduced. Excess Lon is unfavorable to cells under normal conditions. Sonezaki et al. speculated that Lon is normally localized in the nucleoid, which might restrict its ability to access and degrade proteins under normal conditions.27) They also showed that MBP-Lon dissociates from DNA in the presence of denatured but not native bovine serum albumin, and that treatment of MBP-Lon at 47 C reduces its DNA-binding ability but does not affect its protease activity. These findings suggest that exposure to heat-shock causes the release of
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Lon from DNA, allowing it to degrade denatured proteins.27) Similarly, our data suggest that when the levels of polyP increase in response to amino acid starvation, the polyP-Lon complex is released from DNA, whereupon it can access and degrade free ribosomal proteins (Fig. 5). But it might be difficult to maintain that the increase in protein degradation is due only to that of free ribosomal proteins. I assume that the release of Lon to the cytoplasmic space might stimulate the proteolysis of normal proteins in its space, which contributes to the increase in protein degradation under conditions of amino acid starvation. The accumulation of polyP in response to starvation and its ability to promote degradation of substrate proteins by Lon and possibly Clp (K. Nomura, under investigation) provides a model that can explain how cells cope with a sudden depletion of the amino acid pool. This model provides the missing link between protein degradation and the phenomenon known as the ‘‘stringent response.’’ An unexpected protease complex between polyP and Lon is formed during the stringent response, which I propose to call the ‘‘stringent protease.’’ This stringent protease degrades at least free ribosomal proteins, providing amino acids for synthesizing enzymes required for adaptation to a nutrient-poor environment. It appears that polyP is particularly suitable as an adaptor during nutritional downshifts because its synthesis does not require amino acids.
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Acknowledgments I am very grateful to Dr. H. Ohtake for his guidance and continuous support in the studies on polyP. I also thank Dr. A. Kornberg and Dr. J. Kato for helpful discussions. The initial studies on the regulation of polyP accumulation described here were performed in Dr. Kornberg’s laboratory and were supported by the Human Frontier Science Program. The polyP-Lon protease studies were performed with Dr. K. Nomura in Dr. Ohtake’s laboratory. This review was written as a follow-up to the ‘‘Nogei-Kagaku-Shorei award 2004’’ that I received. I would like to thank Dr. T. Miyakawa for recommending me for this award as well as the many students that assisted me in these studies.
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