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 (Ntacetylated) residues. About 90% of human proteins are cotranslationally and irreversibly Nt-acetylated. Many, possiblymost, Nt-acetylated proteins bear N-degrons of the Ac/N-end rule pathway. RATIONALE We wished to identify the recognition component of themultisubunit 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. Themain 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 ofGid4 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 o...

In eukaryotes, N-degron pathways (formerly “N-end rule pathways”) comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal degradation signals called N-degrons, thereby causing degradation of these proteins by the 26S proteasome or autophagy. Gid4, a subunit of the GID ubiquitin ligase in the yeast Saccharomyces cerevisiae, is the recognition component (N-recognin) of the GID-mediated Pro/N-degron pathway. Gid4 targets proteins by recognizing their N-terminal Pro residues or a Pro at position 2, in the presence of distinct adjoining sequence motifs. Under conditions of low or absent glucose, cells make it through gluconeogenesis. When S. cerevisiae grows on a nonfermentable carbon source, its gluconeogenic enzymes Fbp1, Icl1, Mdh2, and Pck1 are expressed and long-lived. Transition to a medium containing glucose inhibits the synthesis of these enzymes and induces their degradation by the Gid4-dependent Pro/N-degron pathway. While studying yeast Gid4, we identified a similar but uncharacterized yeast protein (YGR066C), which we named Gid10. A screen for N-terminal peptide sequences that can bind to Gid10 showed that substrate specificities of Gid10 and Gid4 overlap but are not identical. Gid10 is not expressed under usual (unstressful) growth conditions, but is induced upon starvation or osmotic stresses. Using protein binding analyses and degradation assays with substrates of GID, we show that Gid10 can function as a specific N-recognin of the Pro/N-degron pathway.

Previous studies have shown that translation of mRNA for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions ؊3 to ؊1 relative to the initiation codon. A/A/R (R represents A or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence ؊3 to ؊1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. Although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (؊3 to ؊1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. These results suggest that sequence context is more important for translation initiation in yeast than previously appreciated.

No abstract

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with aKdof 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with aKdof 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeastSaccharomyces cerevisiaecan target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.