An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes

How to remove unnecessary enzymes The terminal amino acid of proteins can determine their longevity. Chen et al. now delineate a third N-end rule pathway that they discovered in yeast. This pathway, termed the Pro/N-end rule pathway, is involved in the turnover of gluconeogenic enzymes after they are no longer required. The authors identified Gid4 as the pathway's Pro/N-recognin and elucidated its specificity for a combination of the N-terminal Pro residue and adjacent sequence motifs in cellular proteins. Science, this issue p. eaal3655 Gid4 is the Pro/N-recognin of a Pro/N-end rule pathway in yeast that targets N-terminal proline and adjacent motifs. INTRODUCTION Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to glucose-replete conditions. Gluconeogenesis (de novo synthesis of glucose) is, in effect, a reversal of glycolysis, in which glucose is converted to pyruvate. Some enzymatic steps are shared between gluconeogenesis and glycolysis, but other steps are confined to one of the two pathways. In the yeast Saccharomyces cerevisiae, the gluconeogenesis-specific enzymes are fructose-1,6-bisphosphatase (Fbp1), isocitrate lyase (Icl1), malate dehydrogenase (Mdh2), and phosphoenolpyruvate carboxykinase (Pck1). We found that Gid4, a subunit of the oligomeric GID ubiquitin ligase, is the recognition component of a proteolytic pathway termed the Pro/N-end rule pathway, which conditionally destroys gluconeogenic enzymes. The N-end rule pathway is a set of proteolytic systems whose unifying feature is their ability to recognize and polyubiquitylate proteins containing N-terminal degradation signals called N-degrons, thereby causing the degradation of these proteins by the proteasome. In eukaryotes, the previously known branches of this system are the Arg/N-end rule pathway and the Ac/N-end rule pathway. The Arg/N-end rule pathway targets specific unacetylated N-terminal residues of cellular proteins, including Asn, Gln, Glu, Asp, Arg, Lys, His, Leu, Phe, Tyr, Trp, Ile, and Met (if Met is followed by a bulky hydrophobic residue). The pathway’s other branch, called the Ac/N-end rule pathway, targets proteins for degradation by recognizing their Nα-terminally acetylated (Nt-acetylated) residues. About 90% of human proteins are cotranslationally and irreversibly Nt-acetylated. Many, possibly most, Nt-acetylated proteins bear N-degrons of the Ac/N-end rule pathway. RATIONALE We wished to identify the recognition component of the multisubunit GID ubiquitin ligase and also to determine whether GID, which was known to mediate the conditional degradation of gluconeogenic enzymes, might recognize them through their N-terminal Pro residues, and also through Pro at position 2. RESULTS The successful strategy involved a version of two-hybrid assay for in vivo protein interactions. The main discovery identified Gid4, a subunit of the GID ubiquitin ligase, as the recognition component (termed Pro/N-recognin) of the Pro/N-end rule pathway. Gid4 was shown to target the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 (and possibly other yeast proteins as well) through the binding to their N-terminal Pro residues in the presence of cognate adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2. Gid4 was also required for the degradation of Pck1 through the ability of Gid4 to target the Pro residue of Pck1 at position 2. The properties of Gid4 discovered so far indicate that its substrate-binding groove can recognize either the N-terminal Pro residue or Pro at position 2 in the presence of cognate adjacent sequence motifs. The recognition flexibility of Gid4 suggests that the true diversity of Gid4 substrates is only beginning to be determined. Subunits of the S. cerevisiae GID ubiquitin ligase have counterparts in animals and plants. Thus, the discovery that yeast Gid4 is the Pro/N-recognin will facilitate the understanding of the Pro/N-end rule pathway in other eukaryotes as well. CONCLUSION With our identification of the Gid4-mediated Pro/N-end rule pathway that specifically recognizes the N-terminal Pro residue, all 20 amino acids of the genetic code have now been shown to act, in specific sequence contexts, as destabilizing N-terminal residues. Thus, most proteins in a cell may be conditionally short-lived N-end rule substrates, either as full-length proteins or as protease-generated natural protein fragments. The Pro/N-end rule pathway and the Gid4 Pro/N-recognin. (Top left) Degradation of the P-Mdh2 (Pro-Mdh2) gluconeogenic enzyme in yeast cells under glucose-replete conditions. (Top right) Two-hybrid protein binding assays with Gid4 vis-à-vis the wild-type P-Fbp1 gluconeogenic enzyme (row 1), its S-Fbp1 mutant (row 2), and analogous pairs of the P-Icl1/S-Icl1 (rows 3 and 4) and P-Pyk2/S-Pyk2 (rows 5 and 6) proteins. Note the binding of Gid4 to P-Fbp1 and P-Icl1. (Bottom) The Pro/N-end rule pathway and the Gid4 Pro/N-recognin. The Gid4 subunit of the multisubunit GID ubiquitin ligase binds to the N-terminal Pro (P) residue of a cellular protein substrate if it also contains a cognate adjoining sequence motif. The targeted substrate is polyubiquitylated and processively destroyed by the 26S proteasome. The same pathway can also recognize substrates in which Pro is at position 2. Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to glucose-replete conditions. We found that the Gid4 subunit of the ubiquitin ligase GID in the yeast Saccharomyces cerevisiae targeted the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 for degradation. Gid4 recognized the N-terminal proline (Pro) residue and the ~5-residue-long adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2; Gid4 directly or indirectly recognized Pro at position 2 of Pck1, contributing to its targeting. These and related results identified Gid4 as the recognition component of the GID-based proteolytic system termed the Pro/N-end rule pathway. Substrates of this pathway include gluconeogenic enzymes that bear either the N-terminal Pro residue or a Pro at position 2, together with adjacent sequence motifs.