RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit

Abstract

The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t 1/2 Ϸ2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4⌬ cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription-activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.proteolysis ͉ ubiquitin ͉ N-end rule ͉ UFD pathway ͉ cell cycle T he Saccharomyces cerevisiae RPN4 gene (its earlier names are SON1 and UFD5) (1) was originally identified through mutant rpn4 alleles that suppressed the growth defect of sec63-101 cells, which bore a temperature-sensitive (ts) variant of SEC63, an essential component of the protein translocation channel in the endoplasmic reticulum membrane (2). More recent studies have shown that mutations in RPN4 inhibit the degradation of normally short-lived proteins that are targeted by the N-end rule pathway, by the ubiquitin͞fusion͞degradation (UFD) pathway, and apparently also by other pathways of the ubiquitin (Ub)-proteasome system (3, 4). These findings suggested that the ability of rpn4 mutations to suppress the conditional lethality of sec63-101 may stem from stabilization of the mutant but partially active SEC63-101 against degradation at nonpermissive temperature.Regulated proteolysis by the Ub͞proteasome system plays essential roles in the cell cycle, differentiation, stress responses, and many other processes (5-7). Ub is a 76-residue protein whose covalent conjugation to other proteins marks these proteins for degradation by the 26S proteasome, an ATP-dependent multisubunit protease. Ub conjugation involves the formation of a thioester between the C terminus of Ub and a specific cysteine of the Ub-activating (E1) enzyme. The Ub moiety of E1ϳUb thioester is transesterified to a cysteine in one of several Ubconjugating (E2) enzymes. The Ub moiety of E2ϳUb thioester is conjugated via the isopeptide bond to the -amino group of either a substrate's Lys residue or a Lys residue of another Ub moiety, the latter reaction resulting in a substrate-linked multi-Ub chain (7,8). Most E2 enzymes function in complexes with proteins called E3 (9-11). The functions of E3s include the initial recognition of degradation signals (degrons) in substrate proteins, with different E3s recognizing different classes of degrons (12)(13)(14). The E2-E3 complexes, referred to as Ub ligases (this term is also used to denote E3s alone), mediate the formation of substrate-linked multi-Ub chains (15, 16). Ubiquitylated substrates are proce...

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