The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons

Abstract

The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates.

Keywords: ATP-dependent Protease; Aat; Adaptor Proteins; ClpA; ClpS; END Site; N-end Rule; Protein Degradation; Protein Processing; Proteomics.

FIGURE 1.

N-end rule substrate immobilization on a ClpS affinity column and elution with peptides possessing N-degrons. Small columns with 1-ml bed volumes of various AminoLink resins were equilibrated with PBS. Samples were loaded, and the columns were washed at ∼1 ml/min with PBS with and without addition of peptides. GFP-proteins were detected by fluorescence measurements. A, LR-GFP_VENUS (1 mg) was applied to a column cross-linked with ClpS. The column was washed with buffer, and 1 mm FKTA was added to elute the bound LR-GFP_VENUS. B, LR-GFP_VENUS (1 mg) was applied to a control resin prepared by inactivation with Tris buffer in the absence of ClpS. Most of the protein (98%) was recovered in the flow-through fractions. No additional protein emerged after FKTA addition. C, LR-GFP_VENUS (1 mg) was added to a ClpS column. The column was washed with buffer containing 1 mm SLRKGE followed by buffer containing 1 mm LRKGE. D, LR-GFP_VENUS (1 mg) was applied to a column cross-linked to the variant ClpS-D35A,D36A. Most of the protein (>95%) was recovered in the flow-through fractions, and no additional protein was detected after adding FKTA.

FIGURE 2.

Differential capture of E. coli cell proteins on a wild-type ClpS affinity column. A, many E. coli proteins bind to wild-type ClpS and not to ClpS_DD/AA. Affinity resins were prepared with either wild-type ClpS or mutated ClpS in which Asp³⁵ and Asp³⁶ were changed to alanine. Extracts of cells harvested during stationary phase were clarified, and equal portions were loaded onto columns (1-ml bed volume) with either wild-type or mutated ClpS. The columns were washed with several column volumes of buffer, and proteins were eluted with buffer containing 1 mm FKTA-NH₂. Fractions of 0.5 ml were collected, and equal aliquots of four fractions that contained protein eluted from the wild-type column were mixed with SDS sample buffer and loaded onto an SDS-polyacrylamide gel (lanes labeled WT). Parallel fractions from the ClpS_DD/AA were loaded in adjacent lanes as indicated (lanes labeled AA). Proteins were detected by staining with Coomassie Blue. B, pulldown of proteins from E. coli cells carrying mutations in the N-end rule pathway. Clarified cell lysates from wild-type, ΔclpSA, or Δaat strains were loaded onto a ClpS affinity column, and bound proteins were eluted with FKTA. No proteins were detected in the FKTA eluate when wild-type lysates were applied to a control column with inactivated (Inact.) resin that had no cross-linked ClpS. Stds, standards.

FIGURE 3.

N-terminal sequencing of the total pool of proteins pulled down by ClpS. Proteins from cell lysates of MG1655 (gray bars) or MG1655 dps⁻ (black bars) were bound and eluted from a ClpS column. They were then precipitated with TCA and dissolved in 4% SDS. The solubilized proteins were spotted on PVDF membranes and after washing the spots with methanol solutions to remove SDS subjected to a single round of Edman sequencing. Individual amino acids are expressed as a percentage of the total amino acids in the first position. The N-degrons, leucine and phenylalanine, are dominant in both samples.

FIGURE 4.

Two-dimensional gel electrophoresis of E. coli proteins eluted from the ClpS affinity column. Cell lysates were prepared from MG1655 (A), MG1655 ΔclpSA (B), and MG1655 aat::kan (C) cells grown to stationary phase in LB medium. The ClpS columns were loaded with 200 mg of protein from clarified cell extracts. Proteins were eluted from the column with FKTA-NH₂, and equal aliquots were treated with TCA to precipitate the protein. Proteins were dissolved in buffered urea and separated by two-dimensional electrophoresis. The total number of protein spots and the relative yields of many individual proteins are both increased in the ΔclpSA strain compared with the parental MG1655 (black circles in panel B). Many proteins are absent or present in lower amounts in pulldowns from cells lacking Aat as well (circled in panel A).

FIGURE 5.

Formation and degradation of the N-end rule fragment of MreB. Wild-type and ΔclpSA cells expressing the MreB-AZ fusion were grown to an A₆₀₀ of 0.8, and chloramphenicol was added to prevent further synthesis. At the times indicated, aliquots of the culture were treated with TCA to stop metabolic and enzymatic activities and to precipitate the protein. After separation by SDS-PAGE, MreB-AZ was detected by Western blotting with HRP-conjugated anti-rabbit IgG, which binds to the AZ domain. A, turnover of MreB-AZ. The full-length fusion protein is indicated as well as the shorter N-end rule substrate that is formed by cleavage at the pro-N-degrons in MreB. The identity of the latter was confirmed in a separate experiment by N-terminal sequencing of the protein isolated on a ClpS affinity column, which was loaded as a reference in the lane marked PD. The cleavage site in the fragment indicated by an asterisk is not known. B, half-life of MreB-AZ in wild-type and clpSA mutant cells. The protein bands detected by Western blotting were scanned, and the protein remaining at each time point was determined from the density. Integration and calculation were performed using the program NIH ImageJ. The full-length protein was cleaved with a half-life of about 1 h in both wild-type (diamonds) and clpSA cells (squares). C, quantitation of the cleaved MreB-AZ protein. The amount of cleaved MreB-AZ at each time was measured by densitometry as above. The N-degron-containing fusion accumulated in the clpSA mutant (25–30% of the original fusion) but not in the wild-type cells. The slight stabilization of the N-end rule form at later times is due to the loss of ClpA, which is unstable and lost from the cells during the chloramphenicol chase.

FIGURE 6.

Model for the generation of N-end rule substrates by partial proteolysis of native proteins. A, pro-N-degron motifs for Aat-independent and -dependent substrates. Examination of the sequences surrounding the pro-N-degron in the N-end rule substrates revealed a possible pattern for sets of Aat-independent and Aat-dependent substrates. Motif 1, for Aat-independent pro-N-degrons, is small-Φ-Φ with the cleavage event occurring between the small and first hydrophobic amino acids (Φ). Motif 2, for Aat-dependent substrates, is Arg-(Lys/Arg) with the cleavage event occurring C-terminal of the first arginine. B, potential locations of pro-N-degrons in native proteins. Natively unstructured regions of proteins or regions that can become exposed are susceptible to cleavage by one or more proteases and peptidases, resulting in the appearance of primary or secondary N-degrons. Modification of the latter by Aat produces a form recognized by ClpS and degraded by ClpAP.