Hai Rao

Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase–anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine—a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 ≈ 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Δ cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Δ cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed theubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.

The Png1-Rad23 complex regulates glycoprotein turnover

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells

The lysosome and 26S proteasome represent the two major proteolytic machines in eukaryotic cells (1). While the lysosome deals mainly with nonselective proteolysis, the 26S proteasome handles the majority of regulated proteolysis. The 26S proteasome is a multisubunit protease that degrades the substrate into small peptides in an ATPdependent manner (2,3). The proteasome has three peptidase activities, including (i) chymotrypsin-like, (ii) trypsin-like, and (iii) peptidylglutamylpeptide hydrolase activities (2). To demonstrate that a protein is a substrate of the proteasome in vivo, the stability of the protein is often examined and compared in the presence or absence of proteasome inhibitors (e.g., MG132), short peptide aldehydes that block active sites of the proteasome (4). Use of the budding yeast Saccharomyces cerevisiae as a model system has been instrumental in uncovering mechanistic attributes and the physiologic functions of the proteasome. However, the use of proteasome inhibitors in wild-type S. cerevisiae cells is hampered by the impermeability of the cell wall or membrane (5). Therefore, mutant yeast strains (e.g., erg6Δ, pdr5Δ) with increased drug permeability or reduced drug efflux are required for experiments using proteasome inhibitors (5,6). A caveat to this approach is that mutation in ERG6 or PDR5 may directly or indirectly affect some cellular processes (e.g., increased import of sodium) (7,8) and protein stability. Furthermore, in some cases, the ERG6 or PDR5 genes must be deleted in another mutant background, a technically cumbersome step, to establish the involvement of the 26S proteasome in a particular process (e.g., transcription or telomere maintenance) (9,10). Recently, a method was developed involving brefeldin A, an antifungal agent often used to study protein trafficking from Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells

Cdc48: A Swiss Army Knife of Cell Biology

Cdc48 (also called VCP and p97) is an abundant protein that plays essential regulatory functions in a broad array of cellular processes. Working with various cofactors, Cdc48 utilizes its ATPase activity to promote the assembly and disassembly of protein complexes. Here, we review key biological functions and regulation of Cdc48 in ubiquitin-related events. Given the broad employment of Cdc48 in cell biology and its intimate ties to human diseases (e.g., amyotrophic lateral sclerosis), studies of Cdc48 will bring significant insights into the mechanism and function of ubiquitin in health and diseases.

Identification of an Htm1 (EDEM)-dependent, Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.

Down-Regulation of Gli Transcription Factor Leads to the Inhibition of Migration and Invasion of Ovarian Cancer Cells via Integrin β4-Mediated FAK Signaling

Background Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including ovarian cancer. Therefore, chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for ovarian cancer. Results In this study, we show that activation of Hh signaling promoted cellular migration and invasion, whereas blockade of Hh signaling with GANT61 suppressed cellular migration and invasion in ovarian cancer cells. After treatment with GANT61, cDNA microarray analyses revealed changes in many genes such as Integrin β4 subunit (ITGB4), focal adhesion kinase (FAK), etc. Furthermore, ITGB4 expression was up-regulated by Sonic Hedgehog (Shh) ligand and down-regulated by Hh signaling inhibitor. The Shh-mediated ovarian cell migration and invasion was blocked by neutralizing antibodies to ITGB4. In addition, phosphorylations of FAK were increased by Shh and decreased by Hh signaling inhibitor. Inhibition of Gli1 expression using siRNA mimicked the effects of GANT61 treatment, supporting the specificity of GANT61. Further investigations showed that activation of FAK was required for Shh-mediated cell migration and invasion. Finally, we found that down-regulation of Gli reduced the expression of ITGB4 and the phosphorylated FAK, resulting in the inhibition of tumor growth in vivo. Conclusions The Hh signaling pathway induces cell migration and invasion through ITGB4-mediated activation of FAK in ovarian cancer. Our findings suggest that the diminishment of crosstalk between phosphorylated FAK and ITGB4 due to the down-regulation of Gli family transcription factors might play a pivotal role for inhibiting ovarian cancer progression.

The Cdc48 ATPase modulates the interaction between two proteolytic factors Ufd2 and Rad23.

Rad23 and cell division cycle protein 48 (Cdc48), two key regulators of postubiquitylation events, act on distinct and overlapping sets of substrates. The principle underlying their division of labor and cooperation in proteolysis remains elusive. Both Rad23 and Cdc48 bind a ubiquitin protein ligase ubiquitin fusion degradation-2 (Ufd2), and regulate the degradation of Ufd2 substrates. With its ability to bind ubiquitin chains directly and the proteasome via different domains, Rad23 serves as a bridge linking ubiquitylated substrates to the proteasome. The significance and specific role of the Ufd2–Cdc48 interaction are unclear. Here, we demonstrate that mutations in Ufd2 alter its interaction with Cdc48 and impair its function in substrate proteolysis but not in ubiquitylation. Furthermore, Cdc48 promotes the disassembly of the Ufd2–Rad23 complex in an manner that is dependent on ATP and Ufd2 binding, revealing a biochemical role for Cdc48. Rad23 was shown to bind separately to Ufd2 and to the proteasome subunit Rpn1, which define two distinct steps in proteolysis. The action of Cdc48 could free Rad23 from Ufd2 to allow its subsequent association with Rpn1, which in turn may facilitate the orderly transfer of the substrate from the ubiquitylation apparatus to the proteasome.

A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2

BackgroundUbiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.ResultsUsing a genome-wide functional screen, we have identified 11 yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins Rad23 and Dsk2. Our results suggest that proper functioning of Rad23 and Dsk2 is required for efficient pheromone response, transcription, amino acid metabolism, and DNA damage response. Two proteins identified by the screen are shown to be proteolytic substrates of Dsk2, validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway.ConclusionIn conclusion, as proof-of-concept, we show that a synthetic dosage lethality screen, which is based on the toxicity induced by gene overexpression, offers an effective, complementary method to elucidating biological functions of proteolytic pathways.

What’s Ub Chain Linkage Got to Do with It?

Ubiquitination—the covalent conjugation of ubiquitin (Ub) to other cellular proteins—regulates a wide range of cellular processes. Often, multiple Ub molecules are added to the substrate to form a Ub chain. Distinct outcomes have been observed for substrates modified with multi-Ub chains linked through particular lysine residues. However, recent studies suggest that Ub chain linkages may not be the key determinant for substrate fate. Here, we review evidence suggesting that Ub-binding proteins play a pivotal role in determining the outcome of substrate ubiquitination. In fulfilling their functions in proteasome-mediated proteolysis or signaling, Ub receptors link ubiquitinated proteins to downstream molecules through protein-protein interactions. Studies of Ub-binding factors may therefore hold the key to understanding the diverse functions of the Ub molecule.