Correction to: Analysis of Cotranslational Polyamine Sensing During Decoding of ODC Antizyme mRNA.

Correction to: Chapter 26 in: Rubén Alcázar and Antonio F. Tiburcio (eds.), Polyamines: Methods and Protocols, Methods in Molecular Biology, vol. 1694, https://doi.org/10.1007/978-1-4939-7398-9_26.

Analysis of Cotranslational Polyamine Sensing During Decoding of ODC Antizyme mRNA.

Polyamines are essential poly-cations with vital functions in all cellular systems. Their levels are controlled by intricate regulatory feedback mechanisms. Abnormally high levels of polyamines have been linked to cancer. A rate-limiting enzyme in the biosynthesis of polyamines in fungi and higher eukaryotes is ornithine-decarboxylase (ODC). Its levels are largely controlled posttranslationally via ubiquitin-independent degradation mediated by ODC antizyme (OAZ). The latter is a critical polyamine sensor in a feedback control mechanism that adjusts cellular polyamine levels. Here, we describe an approach employing quantitative western blot analyses that provides in vivo evidence for cotranslational polyamine-sensing by nascent OAZ in yeast. In addition, we describe an in vitro method to detect polyamine binding by antizyme.

The deubiquitylating enzyme Ubp12 regulates Rad23-dependent proteasomal degradation.

The consecutive actions of the ubiquitin-selective segregase Cdc48 and the ubiquitin shuttle factor Rad23 result in the delivery of ubiquitylated proteins at the proteasome. Here, we show that the deubiquitylating enzyme Ubp12 interacts with Cdc48 and regulates proteasomal degradation of Rad23-dependent substrates in Saccharomyces cerevisiae. Overexpression of Ubp12 results in stabilization of Rad23-dependent substrates. We show that Ubp12 removes short ubiquitin chains from the N-terminal ubiquitin-like domain (UbL) of Rad23. Preventing ubiquitylation of Rad23 by mutation of lysine residues within the UbL domain, Rad23UbLK0, does not affect the non-proteolytic role of Rad23 in DNA repair but causes an increase in ubiquitylated cargo bound to the UBA2 domain of Rad23, recapitulating the stabilization of Rad23-dependent substrates observed upon overexpression of Ubp12. Expression of Rad23UbLK0 or overexpression of Ubp12 impairs the ability of yeast to cope with proteotoxic stress, consistent with inefficient clearance of misfolded proteins by the ubiquitin-proteasome system. Our data suggest that ubiquitylation of Rad23 plays a stimulatory role in the degradation of ubiquitylated substrates by the proteasome.

Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation.

Ubiquitin fusion degradation (UFD) substrates are delivered at the proteasome by a handover mechanism involving the ubiquitin-selective chaperone Cdc48 and the ubiquitin shuttle factor Rad23. Here, we show that introduction of a 20 amino acid peptide extension not only rendered degradation independent of Cdc48, in line with the model that this chaperone is involved in early unfolding events of tightly folded substrates, but at the same time relieved the need for efficient polyubiquitylation and the ubiquitin shuttle factor Rad23. Removal of the ubiquitylation sites in the N-terminal UFD signal made the degradation of this substrate strictly dependent on the peptide extension and also on Cdc48 and, importantly the presence of a functional ubiquitylation machinery. This suggests that the extension in the absence of N-terminal ubiquitylation sites is not properly positioned to engage the unfoldase machinery of the proteasome. Thus the need for efficient ubiquitylation and Cdc48 in facilitating proteasomal degradation are tightly linked but can be bypassed in the context of UFD substrates by the introduction of an unstructured extension. Our data suggest that polyubiquitin-binding complexes acting upstream of the proteasome, rather than the proteasome itself, can be primary determinants for the level of ubiquitylation required for protein degradation.

Polyamines directly promote antizyme-mediated degradation of ornithine decarboxylase by the proteasome.

Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates.

Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA.

Polyamines are essential organic polycations with multiple cellular functions relevant for cell division, cancer and ageing. Regulation of polyamine synthesis is mainly achieved by controlling the activity of ornithine decarboxylase (ODC) through an unusual mechanism involving ODC antizyme, the binding of which disrupts homodimeric ODC and targets it for ubiquitin-independent degradation by the 26S proteasome. Whereas mammals express several antizyme genes, we have identified a single orthologue, termed OAZ1, in Saccharomyces cerevisiae. Similar to its mammalian counterparts, OAZ1 synthesis is induced with rising intracellular polyamine concentrations, which also inhibit ubiquitin-dependent degradation of the OAZ1 protein. Together, these mechanisms contribute to a homeostatic feedback regulation of polyamines. Antizyme synthesis involves a conserved +1 ribosomal frameshifting (RFS) event at an internal STOP codon during decoding of its messenger RNA. Here we used S. cerevisiae OAZ1 to dissect the enigmatic mechanism underlying polyamine regulation of RFS. In contrast with previous assumptions, we report here that the nascent antizyme polypeptide is the relevant polyamine sensor that operates in cis to negatively regulate upstream RFS on the polysomes, where its own mRNA is being translated. At low polyamine levels, the emerging antizyme polypeptide inhibits completion of its synthesis causing a ribosome pile-up on antizyme mRNA, whereas polyamine binding to nascent antizyme promotes completion of its synthesis. Thus, our study reveals a novel autoregulatory mechanism, in which binding of a small metabolite to a nascent sensor protein stimulates the latter's synthesis co-translationally.

The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron.

Ornithine decarboxylase (ODC), a homodimeric enzyme with a rate-limiting function in polyamine biosynthesis, is subject to a feedback control involving its selective proteolysis. Targeting of ODC monomers to the proteasome is mediated by ODC antizyme (OAZ), the expression of which is induced by high levels of polyamines. Here, we report our analysis of the N-terminal degron in Saccharomyces cerevisiae ODC and the mechanism of its antizyme-dependent targeting. This ∼45-residue domain of ODC [termed ODC degradation signal (ODS)] is essential for degradation of ODC. Extensive mutagenesis indicated that it is not a specific sequence within ODS that is important but, rather, its unstructured nature. Consistent with this conclusion, ODS could be functionally replaced by an unrelated unstructured domain. We show that increasing the distance of ODS to the rest of the ODC protein reduced the dependence on Oaz1 for targeting, indicating that exposure of ODS is critical for its function. Disruption of ODC dimers by introducing interface mutations, in contrast, was insufficient for targeting. Binding of Oaz1 to ODC monomers is thus required to activate ODS. Fusion of ODS to the N terminus of Ura3 was sufficient to convert it into a ubiquitin-independent substrate of the proteasome. By contrast, ODS failed to destabilize maltose-binding protein or dihydrofolate reductase, indicating that this degron only operates in an appropriate structural context that enables rapid unfolding.

Hsm3/S5b joins the ranks of 26S proteasome assembly chaperones.

In a recent issue of Molecular Cell, Le Tallec et al. (2009) characterize the yeast Hsm3 protein, an apparent ortholog of human S5b, as a dedicated chaperone that promotes assembly of the base subcomplex of the 19S proteasome activator.

BiP-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes.

Cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) from where it is translocated to the cytosol in a process depending on ATP and luminal ER proteins. To test whether the molecular chaperone BiP (heavy chain binding protein), which is an ER-luminal ATPase, was one of the required proteins the export of CT was analyzed using ER-derived CT-loaded microsomes. The resubstitution of extracted export-incompetent microsomes with purified BiP was sufficient to restore the export of CT. As BiP protected CT from aggregation it is proposed that BiP maintains CT in a soluble, export-competent state.