SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities

Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. Genetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/CDC28. As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE-fractionated yeast extract. Multiubiquitination depends on cyclin/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient to serve as substrate for CDC34-dependent ubiquitination. The complementary C-terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1

Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6

In Saccharomyces cerevisiae, at least 17 DNA replication genes are coordinately expressed at the G_1/S boundary during the cell cycle. All of these genes have the DNA sequence element ACGCGT in their 5' upstream regulatory regions. This sequence has been shown to be essential for periodic expression of the POL1, CDC9, and TMP1 genes. The cyclin (CLN1 and CLN2) and HO genes are another subset of genes that are expressed with the same timing as the DNA replication genes. Their periodic expression requires the participation of two well-characterized transcriptional activators: the SWI4 and SWI6 gene products. In this study, we present evidence that SWI6 contributes to the regulation of DNA replication genes as well. Surprisingly, a preferential requirement for SWI6 over SWI4 is observed in our studies of ACGCGT-dependent reporter gene expression in vivo. This selectivity has not been observed for the other G_1/S genes. Correlating with the in vivo results, protein-DNA complexes formed in vitro on multimeric ACGCGT elements are either abolished or reduced in swi6Δ deletion mutants

Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome

Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97

Components of the ubiquitin-proteasome pathway compete for surfaces on Rad23 family proteins

Background: The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain. Results: In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome. Conclusions: We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting

Development of Protacs to Target Cancer-promoting Proteins for Ubiquitination and Degradation

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein’s function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCFß-TRCP-binding phosphopeptide derived from I{kappa}B{alpha} linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the {alpha} isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer

Cdc48/p97 Mediates UV-Dependent Turnover of RNA Pol II

Cdc48/p97 is an essential ATPase whose role in targeting substrates to the ubiquitin-proteasome system (UPS) remains unclear. Existing models posit that Cdc48 acts upstream of UPS receptors. To address this hypothesis, we examined the association of ubiquitin (Ub) conjugates with 26S proteasomes. Unexpectedly, proteasomes isolated from cdc48 mutants contain high levels of Ub conjugates, and mass spectrometry identified numerous nonproteasomal proteins, including Rpb1, the largest subunit of RNA Pol II. UV-induced turnover of Rpb1 depends upon Cdc48-Ufd1-Npl4, Ubx4, and the uncharacterized adaptor Ubx5. Ubiquitinated Rpb1, proteasomes, and Cdc48 accumulate on chromatin in UV-treated wild-type cells, and the former two accumulate to higher levels in mutant cells, suggesting that degradation of Rpb1 is facilitated by Cdc48 at sites of stalled transcription. These data reveal an intimate coupling of function between proteasomes and Cdc48 that we suggest is necessary to sustain processive degradation of unstable subunits of some macromolecular protein complexes

Charting the protein complexome in yeast by mass spectrometry

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks

Ubistatins Inhibit Proteasome-Dependent Degradation by Binding the Ubiquitin Chain

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys^(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system

Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes

Ribosomal surveillance pathways scan for ribosomes that are transiently paused or terminally stalled owing to structural elements in mRNAs or nascent chain sequences. Some stalls in budding yeast are sensed by the GTPase Hbs1, which loads Dom34, a catalytically inactive member of the archaeo-eukaryotic release factor 1 superfamily. Hbs1–Dom34 and the ATPase Rli1 dissociate stalled ribosomes into 40S and 60S subunits. However, the 60S subunits retain the peptidyl-tRNA nascent chains, which recruit the ribosome quality control complex that consists of Rqc1–Rqc2–Ltn1–Cdc48–Ufd1–Npl4. Nascent chains ubiquitylated by the E3 ubiquitin ligase Ltn1 are extracted from the 60S subunit by the ATPase Cdc48–Ufd1–Npl4 and presented to the 26S proteasome for degradation. Failure to degrade the nascent chains leads to protein aggregation and proteotoxic stress in yeast and neurodegeneration in mice. Despite intensive investigations on the ribosome quality control pathway, it is not known how the tRNA is hydrolysed from the ubiquitylated nascent chain before its degradation. Here we show that the Cdc48 adaptor Vms1 is a peptidyl-tRNA hydrolase. Similar to classical eukaryotic release factor 1, Vms1 activity is dependent on a conserved catalytic glutamine. Evolutionary analysis indicates that yeast Vms1 is the founding member of a clade of eukaryotic release factor 1 homologues that we designate the Vms1-like release factor 1 clade