Formyl-methionine as a degradation signal at the N-termini of bacterial proteins

In bacteria, all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. The fMet residue is cotranslationally deformylated by a ribosome-associated deformylase. The formylation of N-terminal Met in bacterial proteins is not strictly essential for either translation or cell viability. Moreover, protein synthesis by the cytosolic ribosomes of eukaryotes does not involve the formylation of N-terminal Met. What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the similarity of the formyl and acetyl groups, their identical locations in N terminally formylated (Nt formylated) and Nt-acetylated proteins, and the recently discovered proteolytic function of Nt-acetylation in eukaryotes might also signify a proteolytic role of Nt formylation in bacteria. We addressed this hypothesis about fMet based degradation signals, termed fMet/N-degrons, using specific E. coli mutants, pulse-chase degradation assays, and protein reporters whose deformylation was altered, through site-directed mutagenesis, to be either rapid or relatively slow. Our findings strongly suggest that the formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. We suggest that the faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N degrons, in contrast to the cotranslational design of analogous Ac/N degrons in eukaryotes.

. The Arg/N-End Rule Pathway and the Ac/N-End Rule Pathway. N-terminal residues are indicated by single-letter abbreviations. A yellow oval denotes the rest of a protein substrate.
The N-end rule pathway recognizes proteins containing N-terminal degradation signals called Ndegrons, polyubiquitylates these proteins and thereby causes their degradation by the 26S proteasome. Recognition components of the N-end rule pathway are called N-recognins. Eukaryotic N-recognins are E3 ubiquitin (Ub) ligases that can target N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway consists of two branches, described in panels A and B.
(A) The Arg/N-end rule pathway in S. cerevisiae [1][2][3][4][5]. The prefix "Arg" in the pathway's name refers to Nt-arginylation of N-end rule substrates. The Arg/N-end rule pathway targets specific unacetylated N-terminal residues. It is mediated by the Ubr1 N-recognin, a 225 kDa RING-type E3 Ub ligase and a part of the targeting complex containing the Ubr1-Rad6 and Ufd4-Ubc4/5 holoenzymes. The Ubr1 (N-recognin) component of this complex recognizes (binds to) the "primary" destabilizing N-terminal residues Arg, Lys, His, Leu, Phe, Tyr, Trp and Ile, as well as the unmodified N-terminal Met residue, if Met is followed by a bulky hydrophobic (Ф) residue [5]. The terms "secondary" and "tertiary" refer to the indicated enzymatic modifications of specific N-terminal residues. N-terminal Cys can be arginylated by the Ate1 arginyltransferase (R-transferase) only after the oxidation of Cys to Cys-sulfinate or Cys-sulfonate, in reactions that involve nitric oxide (NO) and oxygen [2-4, 6, 7]. Regulated oxidation of N-terminal Cys takes place in multicellular eukaryotes but not in fungi such as S. cerevisiae, which apparently do not produce NO under normal conditions.

(B)
The Ac/N-end rule pathway in S. cerevisiae [3,5,[8][9][10]. This pathway recognizes substrates through their N α -terminally acetylated (Nt-acetylated) residues. The corresponding degradation signals and E3 Ub ligases are called Ac/N-degrons and Ac/N-recognins, respectively. Red arrow on the left indicates the removal of N-terminal Met by Met-aminopeptidases (MetAPs). Nterminal Met is retained if a residue at position 2 is larger than Val [11][12][13]. The term "secondary" refers to the requirement for a modification (Nt-acetylation) of a destabilizing N-terminal residue before a protein can be recognized by an Ac/N-recognin.
This study