Degradation Of N-end Rule Substrates Research Articles

Characterization of ClpS2, an essential adaptor protein for the cyanobacterium Synechococcus elongatus

The adaptor protein ClpS associates to the Clp protease and promotes degradation of N-end rule substrates in eubacteria and in algal/plant chloroplasts. Cyanobacteria are unusual in having two distinct ClpS paralogs. Although ClpSl is typical of bacterial ClpS, ClpS2 differs in crucial ways. ClpS2 in Synechococcus elongatus is a relatively low-abundant, soluble protein essential for phototrophic growth. Like ClpSl, ClpS2 binds to the ClpCP3/R protease to block α-casein degradation and promote that of N-end rule substrates in vitro. However, their substrate specificity differs, with ClpSl recognizing destabilizing Phe and Tyr residues at the substrate N-terminus whereas ClpS2 recognizes Leu. Overall, ClpS2 appears to have independently evolved in cyanobacteria to degrade a particular group of proteins, whose turnover is vital for cell viability.

Role of N‐end rule ubiquitin ligases UBR1 and UBR2 in regulating the leucine‐mTOR signaling pathway

Of 20 natural amino acids, leucine is particularly important for promoting cellular protein synthesis. The effect of leucine involves mammalian target of rapamycin (mTOR), a key protein kinase controlling cell growth. Leucine enhances mTOR-mediated phosphorylation of S6K1 and 4E-BP, thereby promoting protein synthesis. However, how the presence of leucine is sensed and transmitted to mTOR is poorly understood. Here, we show evidence that UBR1 and UBR2 might be cellular targets of leucine. UBR1 and UBR2 are E3 ubiquitin ligases that recognize the identity of N-terminal residues and contribute to selective destabilization of target proteins according to the N-end rule. Using leucine-immobilized affinity beads, we identified UBR1 and UBR2 as leucine-binding proteins from leucine-responsive rat hepatoma H4IIE cells. Over-expression of UBR1 or UBR2 resulted in a reduction in mTOR-dependent S6K1 phosphorylation, whereas knockdown of UBR1 or UBR2 increased S6K1 phosphorylation in amino acid-starved human 293T cells. We also found that leucine binds to the substrate-recognition domain of UBR2 and inhibits degradation of N-end rule substrates in vitro. These findings suggest that UBR1 and UBR2 are negative regulators of the leucine-mTOR signaling pathway. Leucine might activate this pathway in part through inhibition of their ubiquitin ligase activity.

Modification of PATase by L/F-transferase generates a ClpS-dependent N-end rule substrate in Escherichia coli

The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing an N-degron are recognised by ClpS and degraded by ClpAP in an ATP-dependent manner. Here, we report the isolation of 23 ClpS-interacting proteins from E. coli. Our data show that at least one of these interacting proteins--putrescine aminotransferase (PATase)--is post-translationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl-tRNA-protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N-terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N-end rule pathway, through the ClpAPS-mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N-degron, which is required for substrate delivery to ClpA.

ClpS modulates but is not essential for bacterial N-end rule degradation

In eubacteria, the ClpS adaptor has been proposed to be essential for degradation of N-end rule substrates by the AAA(+) protease ClpAP. To test this model, we assayed degradation of substrates bearing N-end rule sequences isolated in a genetic screen for efficient degradation tags. ClpS was not vital for degradation in vivo but rather stimulated turnover in a sequence-specific manner. Although ClpS substantially enhanced degradation of N-end substrates at low substrate concentrations in vitro, it suppressed the degradation rate when substrate was saturating. Thus, we conclude that ClpAP recognizes N-end rule substrates directly, whereas ClpS modulates this degradation pathway.

ClpS is an essential component of the N-end rule pathway in Escherichia coli

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS-ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.

Intracellularly inducible, ubiquitin hydrolase-insensitive tandem ubiquitins inhibit the 26S proteasome activity and cell division.

We constructed polyubiquitin derivatives that contain a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. They were designated tandem ubiquitin (tUb) with the number of repeats, such as tUb2. When tUbs were expressed under the control of the GAL1 promoter in the wild-type yeast strain, growth was strongly inhibited. Under these conditions, the degradation of N-end rule substrates, a UFD substrate and Gcn4 was inhibited, indicating that the tUb inhibits 26S proteasome activity. Consistent with this, tUb binds to the 26S proteasome. We showed that tUb inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.

PRT1 of Arabidopsis is a ubiquitin protein ligase of the plant N-end rule pathway with specificity for aromatic amino-terminal residues.

The gene PRT1 of Arabidopsis, encoding a 45-kD protein with two RING finger domains, is essential for the degradation of F-dihydrofolate reductase, a model substrate of the N-end rule pathway of protein degradation. We have determined the function of PRT1 by expression in yeast (Saccharomyces cerevisiae). PRT1 can act as a ubiquitin protein ligase in the heterologous host. The identified substrates of PRT1 have an aromatic residue at their amino-terminus, indicating that PRT1 mediates degradation of N-end rule substrates with aromatic termini but not of those with aliphatic or basic amino-termini. Expression of model substrates in mutant and wild-type plants confirmed this substrate specificity. A ligase activity exclusively devoted to aromatic amino-termini of the N-end rule pathway is apparently unique to plants. The results presented also imply that other known substrates of the plant N-end rule pathway are ubiquitylated by one or more different ubiquitin protein ligases.

Degradation of ornithine decarboxylase in Saccharomyces cerevisiae is ubiquitin independent

Ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine biosynthesis is one of the most rapidly degraded proteins in eukaryotic cells. Mammalian ODC is a notable exception to the widely accepted dogma that ubiquitination is always required for targeting a protein to degradation by the 26S proteasome. However, while it is well established that in mammalian cells degradation of ODC is ubiquitin independent, the requirement of ubiquitination for degradation of ODC in yeast cells remained undetermined. We have investigated ODC degradation in three mutant strains of Sacharomyces cerevisiae in which ubiquitin-dependent protein degradation activity is severely compromised. While yeast ODC was rapidly degraded in all these mutant strains the degradation of N-end rule substrates was inhibited. A mutant mouse ODC that fails to interact with Az was rapidly degraded in yeast cells but was stable in mammalian cells suggesting that interaction with a mammalian Az like yeast protein is not necessary for the degradation of ODC in yeast cells. Deletion analysis revealed that sequences from its unique N-terminus are involved in targeting yeast ODC to rapid degradation in yeast cells.

The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain.

We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p. However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates. In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system.

Substrate Recognition by the Leucyl/Phenylalanyl-tRNA-protein Transferase: CONSERVATION WITHIN THE ENZYME FAMILY AND LOCALIZATION TO THE TRYPSIN-RESISTANT DOMAIN

The leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) from Escherichia coli catalyzes a peptidyltransferase reaction that results in the N-terminal aminoacylation of acceptor proteins using Leu-, Phe-, and Met-tRNAs as amino acid donors. We demonstrated that L/F-transferase homologs are widely distributed throughout the eubacteria, supporting our proposal that the enzyme family is ancient and catalyzes early peptide bond synthesis. However, here we present data suggesting that the L/F-transferase is not a homolog of the peptidyltransferase enzymes involved in cell wall peptidoglycan biosynthesis in Gram-positive species, such as Staphylococcus aureus. A sequence comparison of the known L/F-transferase homologs began to identify the essential residues required to catalyze a peptidyltransferase reaction and revealed that <20% of the residues were invariant within the L/F-transferase family. Despite this sequence variation, substrate specificity was broadly conserved, and L/F-transferase homologs from Providencia stuartii, Vibrio cholerae, Neisseria gonorrhoeae, and the cyanobacterium Synechocystis sp. all complemented an E. coli aat mutant (lacking L/F-transferase activity) for the degradation of N-end rule substrates. In vitro comparison of the most divergent L/F-transferase homologs, from E. coli and the cyanobacterium Synechocystis sp., revealed near-complete conservation of both substrate specificity and secondary structure. Finally, we demonstrated that variants of the E. coli L/F-transferase, lacking either 33 or 78 N-terminal residues, retained measurable peptidyltransferase activity and wild type substrate specificity. Overall, our results identified an essential core of an L/F-transferase and revealed that a peptidyltransferase catalyst may be constructed from approximately 120 amino acids.

Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.

The N-End Rule in Bacteria

The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all eukaryotes examined. It is shown that the bacterium Escherichia coli also has the N-end rule pathway. Amino-terminal arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan confer 2-minute half-lives on a test protein; the other amino-terminal residues confer greater than 10-hour half-lives on the same protein. Amino-terminal arginine and lysine are secondary destabilizing residues in E. coli because their activity depends on their conjugation to the primary destabilizing residues leucine or phenylalanine by leucine, phenylalanine-transfer RNA-protein transferase. The adenosine triphosphate-dependent protease Clp (Ti) is required for the degradation of N-end rule substrates in E. coli.