Role of the San1 ubiquitin ligase in the heat stress-induced degradation of nonnative Nup1 in the nuclear pore complex.

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex.

Defective import of mitochondrial metabolic enzyme elicits ectopic metabolic stress.

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCF.

Metabolomics analysis of an AAA-ATPase Cdc48-deficient yeast strain.

The ubiquitin-specific chaperone AAA-ATPase Cdc48 and its orthologs p97/valosin-containing protein (VCP) in mammals play crucial roles in regulating numerous intracellular pathways via segregase activity, which separates polyubiquitinated targets from membranes or binding partners. Interestingly, high-throughput experiments show that a vast number of metabolic enzymes are modified with ubiquitin. Therefore, Cdc48 may regulate metabolic pathways, for example by acting on the polyubiquitin chains of metabolic enzymes; however, the role of Cdc48 in metabolic regulation remains largely unknown.

Triacylglycerol lipase Tgl4 is a stable protein and its dephosphorylation is regulated in a cell cycle-dependent manner in Saccharomyces cerevisiae.

Triacylglycerols (TGs) serve as reservoirs for diacylglycerols and fatty acids, which play important roles in synthesizing energy and membrane lipids that are required for cell cycle progression. In the yeast, Saccharomyces cerevisiae, Tgl4, the functional ortholog of murine adipose triacylglycerol lipase (ATGL), is activated by Cdk1/Cdc28-mediated phosphorylation and facilitates the G1/S transition. However, little is known about how Tgl4 is inactivated during the cell cycle.

Proteolysis of adaptor protein Mmr1 during budding is necessary for mitochondrial homeostasis in Saccharomyces cerevisiae.

In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae.

Dot6/Tod6 degradation fine-tunes the repression of ribosome biogenesis under nutrient-limited conditions.

Ribosome biogenesis (Ribi) is a complex and energy-consuming process, and should therefore be repressed under nutrient-limited conditions to minimize unnecessary cellular energy consumption. In yeast, the transcriptional repressors Dot6 and Tod6 are phosphorylated and inactivated by the TORC1 pathway under nutrient-rich conditions, but are activated and repress ∼200 Ribi genes under nutrient-limited conditions. However, we show that in the presence of rapamycin or under nitrogen starvation conditions, Dot6 and Tod6 were readily degraded by the proteasome in a SCF and Tom1 ubiquitin ligase-dependent manner, respectively.

A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed.

ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation.

Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell-cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis.

Potential Physiological Relevance of ERAD to the Biosynthesis of GPI-Anchored Proteins in Yeast.

Misfolded and/or unassembled secretory and membrane proteins in the endoplasmic reticulum (ER) may be retro-translocated into the cytoplasm, where they undergo ER-associated degradation, or ERAD. The mechanisms by which misfolded proteins are recognized and degraded through this pathway have been studied extensively; however, our understanding of the physiological role of ERAD remains limited. This review describes the biosynthesis and quality control of glycosylphosphatidylinositol (GPI)-anchored proteins and briefly summarizes the relevance of ERAD to these processes.

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.

The UGA codon signals protein translation termination, but it can also be translated into selenocysteine (Sec, U) to produce selenocysteine-containing proteins (selenoproteins) by dedicated machinery. As Sec incorporation can fail, Sec-containing longer and Sec-lacking shorter proteins co-exist. Cul2-type ubiquitin ligases were recently shown to destabilize such truncated proteins; however, which ubiquitin ligase targets truncated proteins for degradation remained unclear.

Msp1 Clears Mistargeted Proteins by Facilitating Their Transfer from Mitochondria to the ER.

Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tail-anchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER).

Harmonizing Experimental Data with Modeling to Predict Membrane Protein Insertion in Yeast.

Membrane proteins must adopt their proper topologies within biological membranes, but achieving the correct topology is compromised by the presence of marginally hydrophobic transmembrane helices (TMHs). In this study, we report on a new model membrane protein in yeast that harbors two TMHs fused to an unstable nucleotide-binding domain. Because the second helix (TMH2) in this reporter has an unfavorable predicted free energy of insertion, we employed established methods to generate variants that alter TMH2 insertion free energy.

Heterologous expression and functional analysis of the F-box protein Ucc1 from other yeast species in Saccharomyces cerevisiae.

The ubiquitin-proteasome system plays an important role in metabolic regulation. In a previous study, we reported that, in Saccharomyces cerevisiae, when glucose is available, the SCF ubiquitin ligase complex targets citrate synthase 2 (Cit2) for proteasomal degradation, thereby suppressing the glyoxylate cycle, an anabolic pathway that replenishes the TCA cycle with succinate for the activation of gluconeogenesis. However, the roles of Ucc1 in other yeast species remain unclear.

Hepatic Sdf2l1 controls feeding-induced ER stress and regulates metabolism.

Dynamic metabolic changes occur in the liver during the transition between fasting and feeding. Here we show that transient ER stress responses in the liver following feeding terminated by Sdf2l1 are essential for normal glucose and lipid homeostasis. Sdf2l1 regulates ERAD through interaction with a trafficking protein, TMED10.

The HECT-type ubiquitin ligase Tom1 contributes to the turnover of Spo12, a component of the FEAR network, in G2/M phase.

The ubiquitin-proteasome system plays a crucial role in cell cycle progression. A previous study suggested that Spo12, a component of the Cdc14 early anaphase release (FEAR) network, is targeted for degradation by the APC/C complex in G1 phase. In the present study, we demonstrate that the Hect-type ubiquitin ligase Tom1 contributes to the turnover of Spo12 in G2/M phase.

Ubiquitin ligase SPSB4 diminishes cell repulsive responses mediated by EphB2.

Eph receptor tyrosine kinases and their ephrin ligands are overexpressed in various human cancers, including colorectal malignancies, suggesting important roles in many aspects of cancer development and progression as well as in cellular repulsive responses. The ectodomain of EphB2 receptor is cleaved by metalloproteinases (MMPs) MMP-2/MMP-9 and released into the extracellular space after stimulation by its ligand. The remaining membrane-associated fragment is further cleaved by the presenilin-dependent γ-secretase and releases an intracellular peptide that has tyrosine kinase activity.

Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates.

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum-associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood.

Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways.

ASB7 regulates spindle dynamics and genome integrity by targeting DDA3 for proteasomal degradation.

Proper dynamic regulation of the spindle is essential for successful cell division. However, the molecular mechanisms that regulate spindle dynamics in mitosis are not fully understood. In this study, we show that Cullin 5-interacting suppressor of cytokine signaling box protein ASB7 ubiquitinates DDA3, a regulator of spindle dynamics, thereby targeting it for proteasomal degradation.

Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α.

pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a ubiquitin ligase that targets hypoxia-inducible factor α (HIF-α) for proteasomal degradation. Although HIF-α activation is necessary for VHL disease pathogenesis, constitutive activation of HIF-α alone did not induce renal clear cell carcinomas and pheochromocytomas in mice, suggesting the involvement of an HIF-α-independent pathway in VHL pathogenesis. Here, we show that the transcription factor B-Myb is a pVHL substrate that is degraded via the ubiquitin-proteasome pathway and that vascular endothelial growth factor (VEGF)- and/or platelet-derived growth factor (PDGF)-dependent tyrosine 15 phosphorylation of B-Myb prevents its degradation.

The role of cullin 5-containing ubiquitin ligases.

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation.

Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.

During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast.

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes.

The Ubiquitin Ligase SCF(Ucc1) Acts as a Metabolic Switch for the Glyoxylate Cycle.

Despite the crucial role played by the glyoxylate cycle in the virulence of pathogens, seed germination in plants, and sexual development in fungi, we still have much to learn about its regulation. Here, we show that a previously uncharacterized SCF(Ucc1) ubiquitin ligase mediates proteasomal degradation of citrate synthase in the glyoxylate cycle to maintain metabolic homeostasis in glucose-grown cells. Conversely, transcription of the F box subunit Ucc1 is downregulated in C2-compound-grown cells, which require increased metabolic flux for gluconeogenesis.