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

Abstract

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...

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

“…Gid4, a subunit of GID that functions as the Pro/N-recognin, targets proteins that bear the N-terminal Pro residue or a Pro at position 2, in addition to adjoining (and also required) sequence motifs ( Fig. 1C) (21). Physiological substrates of the Pro/N-end rule pathway include gluconeogenic enzymes, which are long-lived in cells deprived of glucose but are selectively destroyed upon return to glucose-replete conditions (21,51,52).…”

“…1C) (21). Physiological substrates of the Pro/N-end rule pathway include gluconeogenic enzymes, which are long-lived in cells deprived of glucose but are selectively destroyed upon return to glucose-replete conditions (21,51,52).…”

“…1) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The main determinant of an N-degron is a destabilizing Nt-residue of a protein.…”

“…Studies over the last three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing Nt-residues. Consequently, many, possibly most, proteins in a cell are conditionally short-lived N-end rule substrates, either as full-length proteins or as protease-generated natural protein fragments (21,27). Recognition components of N-end rule pathways are called "N-recognins."…”

Group VII Ethylene Response Factors in Arabidopsis: Regulation and Physiological Roles

“…These proteins modulate the autophagy pathway and thereby enhance Arabidopsis tolerance to nutrient starvation (Qi et al, 2017). In fact, autophagy responses are also activated during hypoxia and contribute to plant submergence tolerance (Chen et al, 2015(Chen et al, , 2017a(Chen et al, and 2017b. This evidence suggests that an additional tier of regulation might connect the ERF-VIIs to submergence responses, through the SINAT factors.…”