Role of Inorganic Polyphosphate in Promoting Ribosomal Protein Degredation by the Lon Protease in E. coli Akio Kuroda, Kazutaka Nomura, Ryo Ohtomo, Junichi Kato, Tsukasa Ikeda, Noboru Takiguchi, Hisao Ohtake, Arthur Kornberg

Ribosomal proteins separated by SDS-PAGE were transferred to nitrocellulose membrane (pore size 0.2m, Schleicher & Schuell). Membranes were blocked with 5% BSA in a binding buffer (50mM Tris-Cl pH 7.5, 50mM NaCl) for overnight at 4°C and then blotted with the binding buffer containing ³²P-polyP (0.2Ci/ml) for 60 min at 4°C. Membranes were washed with the binding buffer, dried, and visualized by Phospho-Imager. Some of ribosomal proteins were identified by Toff mass spectroscopy. Most substrates for the Lon-polyP system are basic ribosomal proteins that can bind to polyP. However, proteins that can bind to polyP are not always degraded; for instance, polyP kinase that binds to polyP is not well degraded by Lon and polyP. PolyP did not stimulate Lon-dependent degradation of MBP-sulA or casein.

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Supplemental Figure 2. Degradation of intact ribosomes with Lon and polyP.

Intact ribosomes (7.3g) were incubated with MBP-Lon (3.6g) in the presence or absence of polyP (1.6 M) and RNase (0.3 g) in a 60-l reaction mixture at 37 °C. All reaction mixtures contained 1 mM ATP, 20 mM Tris-HCl [pH 7.4], and 5 mM MgCl₂. At the indicated times, 14 l were removed from the reaction mixture, and the reaction was stopped by mixing with SDS-buffer. Ribosomes treated with RNase can still be precipitated by centrifugation, thus it is likely that the ribosome is only partially altered in this condition. PolyP used in these experiments is the long-chain polyP (700 P_i residues). We tested whether polyP inhibits in vitro protein translation using an S30 fraction. No inhibition of ³⁵S-methionine incorporation was observed in the presence of polyP at 1.4 M. These results suggest that polyP alone does not stimulate disassembly of the ribosome, but polyP and Lon degrade ribosomal proteins in the RNase-distorted ribosome.

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