R. Jürgen Dohmen

UMP1P IS REQUIRED FOR PROPER MATURATION OF THE 20S PROTEASOME AND BECOMES ITS SUBSTRATE UPON COMPLETION OF THE ASSEMBLY

We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process. The S. cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed beta subunits but is not detected in the mature 20S proteasome. Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. Cells lacking Ump1p exhibit a lack of coordination between the processing of beta subunits and proteasome assembly, resulting in functionally impaired proteasomes. We also show that the propeptide of the Pre2p/Doa3p beta subunit is required for Ump1ps function in proteasome maturation.

Ubiquitin-dependent Proteolytic Control of SUMO Conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.

SUMO playing tag with ubiquitin

In addition to being structurally related, the protein modifiers ubiquitin and SUMO (small ubiquitin-related modifier), share a multitude of functional interrelations. These include the targeting of the same attachment sites in certain substrates, and SUMO-dependent ubiquitylation in others. Notably, several cellular processes, including the targeting of repair machinery to DNA damage sites, require the sequential sumoylation and ubiquitylation of distinct substrates. Some proteins promote both modifications. By contrast, the activity of some enzymes that control either sumoylation or ubiquitylation is regulated by the respective other modification. In this review, we summarize recent findings regarding intersections between SUMO and ubiquitin that influence genome stability and cell growth and which are relevant in pathogen resistance and cancer treatment.

SUMO-targeted ubiquitin ligases.

Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate‐limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin‐independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine‐induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine‐regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin‐mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC‐mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.

Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML

We have recently reported that poly‐SUMO‐2/3 conjugates are subject to a ubiquitin‐dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO‐2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMO‐binding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO. Reconstitution of SUMO‐dependent ubiquitylation of PML by RNF4 in vitro and in a yeast trans vivo system revealed a preference of RNF4 for chain forming SUMOs. Polysumoylation of PML in response to ATO thus leads to its recognition and ubiquitylation by RNF4.

Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome

Analysis of several Saccharomyces cerevisiae ump mutants with defects in ubiquitin (Ub)‐mediated proteolysis yielded insights into the regulation of the polyubiquitin gene UBI4 and of proteasome genes. High‐molecular weight Ub–protein conjugates accumulated in ump mutants with impaired proteasome function with a concomitant decrease in the amount of free Ub. In these mutants, transcriptional induction of UBI4 was depending in part on the transcription factor Rpn4. Deletion of UBI4 partially suppressed the growth defects of ump1 mutants, indicating that accumulation of polyubiquitylated proteins is deleterious to cell growth. Transcription of proteasome subunit genes was induced in ump mutants affecting the proteasome, as well as under conditions that mediate DNA damage or the formation of abnormal proteins. This induction required the transcriptional activator Rpn4. Elevated Rpn4 levels in proteasome‐deficient mutants or as a response to abnormal proteins were due to increased metabolic stability. Up‐regulation of proteasome genes in response to DNA damage, in contrast, is shown to operate via induction of RPN4 transcription.

The C-terminal Extension of the β7 Subunit and Activator Complexes Stabilize Nascent 20 S Proteasomes and Promote Their Maturation

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. β7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the α ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in β subunit maturation. The effect of blm10Δ on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4 C terminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.

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.

Heat-inducible degron and the making of conditional mutants.

Conditional mutants retain the function of a specific gene under one set of conditions, called permissive, and lack that function under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild-type allele of a gene. Such mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature-sensitive (ts) mutants, first used in functional studies more than half a century ago, remain a mainstay of genetic analyses. One limitation of the classical ts approach is the uncertainty as to whether a given gene can be mutated to yield a ts product. Another problem with conventional ts mutations is that they are often too leaky to be useful. In 1994, we described a new method, based on a heat-activated degradation signal (degron) that is targeted by the N-end-rule pathway in the yeast Saccharomyces cerevisiae. The corresponding mutants were termed td (temperature-activated degron) to distinguish them from conventional ts mutants. The td method requires neither a missense mutation in a gene of interest nor an alteration in its expression patterns. Arg-DHFR(ts), a ts variant of dihydrofolate reductase-bearing N-terminal Arg residue (a destabilizing residue in the N-end rule) was shown to function as a portable, heat-activated degron, in that Arg-DHFR(ts) was long-lived at 23 degrees but became short-lived at 37 degrees , owing to activation of its previously cryptic degron. Linking, in a linear fusion, this portable ts-degron to a protein of interest results in destruction of the latter at 37 degrees , thereby yielding a ts (td) mutant of a corresponding gene. Since the introduction of the td method in 1994, numerous studies have successfully used td alleles of specific genes in functional analyses.