Abstract
Chaperone proteins help other proteins to fold. GroEL, the Escherichia coli form of the ubiquitous Cpn60 chaperonins, has a multimeric barrel-shaped structure with a central cavity, within which almost any protein can fold in vitro1. But what does GroE (GroEL plus its co-chaperone GroES) fold in the cell? Why is it needed for cell survival2? We report here the first definite identification of an essential, GroE-dependent E. coli protein, dihydropicolinate synthase (DapA), without which cell-wall synthesis fails.
Main
Although GroE is abundant, calculation3 suggests there is enough in a cell to interact with only about 10% of newly synthesized proteins. While several hundred different species of newly synthesized protein transiently associate with GroE, they comprise fewer than 20% of possible types4, only a minority of which are likely to be essential.
We find that cells deprived of GroE lyse because they fail to make cell walls. Lysis is due to a lack of the cell-wall precursor diaminopimelic acid (DAP), which fails to be made because the level of DapA — the first enzyme in the DAP-synthesis pathway — is greatly reduced in GroE-deficient cells.
When GroE levels were reduced5 or a mutant form of the protein inactivated by temperature shift6, changes in the levels of many proteins were noted, but no particular protein was identified as both GroE-dependent and essential for cell survival. By replacing the native groE chromosomal promoter region with the araC gene and araBAD promoter (Fig. 1a), we created a strain dependent on added arabinose for continued viability and GroE production, and were able to use it to determine the cause of death as GroE is depleted.
Following arabinose removal, GroE levels halve with each generation (Fig. 1b). Growth remains exponential for about 2 hours (Fig. 2a; GroE level will be reduced to 2-3% of normal by this time) and then continues at a decreasing rate for another 1.5 hours; the cells then abruptly lyse. That cells can continue to grow exponentially with greatly reduced levels of GroE suggests either that any essential GroE-dependent proteins are present in considerable excess, or that they can effectively co-opt any GroE available3.
During GroE depletion in osmotically protected medium, the cells are converted to spherical protoplasts, indicating a defect in synthesis or maintenance of the peptidoglycan cell wall. We followed peptidoglycan synthesis by measuring the rate of incorporation of [3H]DAP, a specific precursor, into peptidoglycan7. Uptake was greatly enhanced in GroE- depleted cells, suggesting that these cells might be DAP-starved.
In confirmation of this, we found that addition of DAP to the growth medium (Fig. 2a) prevents cell lysis and permits growth to continue for up to 6 hours. Furthermore, although addition of DAP to solid medium does not support colony formation — confirming that GroE is essential for processes other than cell wall synthesis — when DAP is present, colony formation requires less arabinose than is normally the case. This suggests that at least a part of the cellular complement of GroE is dedicated to ensuring that DAP is available, and that GroE is likely to have a role in maintaining the levels of an enzyme or enzymes involved in DAP synthesis.
DAP is synthesized from aspartic semialdehyde in six steps8. Deficiency of any of the enzymes DapA-E causes cell lysis9. We reasoned that if a critical enzyme were to be synthesized in excess before GroE depletion, lysis would be delayed. We cloned dapA, B, D and E separately into pBR325 to achieve overproduction of the encoded enzymes. The presence of the dapA, but not of the other dap plasmids, delays lysis by several hours (Fig. 2a), suggesting that levels of DapA may be influenced by GroE. Immediately before lysis, the level of DapA in the depleted cells is reduced to 16% of normal (Fig. 2b). The remaining protein is soluble and stable. In contrast, the levels of several other essential proteins examined do not decrease during GroE depletion.
Thus we see that levels of an essential protein, DapA, are dependent on GroE. We suggest that decreased stability of nascent DapA, as a consequence of failure of GroE-assisted folding, is the most likely reason for DapA deficiency.
References
Martin, J. & Hartl, F. U. Curr. Opin. Struct. Biol. 7, 41–52 (1997).
Fayet, O., Ziegelhoffer, T. & Georgopolous, T. C. J. Bacteriol. 171, 1379–1385 (1989).
Lorimer, G. H. FASEB J. 10, 5–9 (1996).
Ewalt, K. L. et. al. Cell 90, 491–500 (1997).
Kanemori, M., Mori, H. & Yura, T. J. Bacteriol. 176, 4235–4242 (1994).
Horwich, A. L. et al. Cell 74, 909–917 (1993).
Höltje, J. V. et al. J. Bacteriol. 124, 1067–1076 (1975).
Cohen, G. N. in Amino Acids: Biosynthesis and Genetic Regulation (eds Herrmann, K. M. & Somerville, R. L.) 147-171 (Addison-Wesley, Reading, MA., 1983).
Patte, J.-C. in Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn (ed. Neidhardt, F. C.) 528-541 (ASM, Washington DC, 1996).
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McLennan, N., Masters, M. GroE is vital for cell-wall synthesis. Nature 392, 139 (1998). https://doi.org/10.1038/32317
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DOI: https://doi.org/10.1038/32317
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