Regulation of the Gid ubiquitin ligase recognition subunit Gid4.

Glucose consumption via glycolysis and its biosynthesis via gluconeogenesis are central reciprocal pathways controlled by a set of different enzymes. In the yeast Saccharomyces cerevisiae, expression of gluconeogenic enzymes is induced when cells are devoid of glucose. Availability of glucose immediately leads to inactivation and rapid degradation of these enzymes via the ubiquitin proteasome system. Polyubiquitination is carried out by the Gid complex, a multisubunit RING E3 ligase that constitutively consists of six different proteins. Upon addition of glucose to the medium, the substrate recognition subunit Gid4 appears within minutes and triggers ubiquitination of the gluconeogenic enzymes. Here, we show that Gid4 is tightly regulated on the transcriptional and protein level to ensure proper adjustment of gluconeogenesis.

Mechanisms of cell regulation - proteolysis, the big surprise.

Precise regulation of cellular processes is essential for life. Regarding proteins, many regulatory mechanisms were explored over the years, such as posttranslational modifications (e.g., phosphorylation), enzyme activation or inhibition by small molecules, and modulation of protein-protein interactions. Complete removal of a protein via proteolysis as a regulatory mechanism, however, was denied for a long time, mainly due to economical considerations. Scientists could not believe that a protein which is synthesized at the expense of a lot of energy could be destroyed again. Here, we discuss the landmark discoveries and the use of yeast as a eukaryotic model organism that finally paved the way for our current understanding of proteolysis as an essential regulatory principle in the cell.

Protein Quality Control of the Endoplasmic Reticulum and Ubiquitin-Proteasome-Triggered Degradation of Aberrant Proteins: Yeast Pioneers the Path.

Cells must constantly monitor the integrity of their macromolecular constituents. Proteins are the most versatile class of macromolecules but are sensitive to structural alterations. Misfolded or otherwise aberrant protein structures lead to dysfunction and finally aggregation. Their presence is linked to aging and a plethora of severe human diseases. Thus, misfolded proteins have to be rapidly eliminated. Secretory proteins constitute more than one-third of the eukaryotic proteome. They are imported into the endoplasmic reticulum (ER), where they are folded and modified. A highly elaborated machinery controls their folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol. In the cytosol, they are degraded by the highly selective ubiquitin-proteasome system. This process of protein quality control followed by proteasomal elimination of the misfolded protein is termed ER-associated degradation (ERAD), and it depends on an intricate interplay between the ER and the cytosol.

Characterization of protein quality control components via dual reporter-containing misfolded cytosolic model substrates.

Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and ß-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast.

Molecular mass as a determinant for nuclear San1-dependent targeting of misfolded cytosolic proteins to proteasomal degradation.

Most misfolded cytosolic proteins in the cell are eliminated by the ubiquitin-proteasome system. In yeast, polyubiquitination of misfolded cytosolic proteins is triggered mainly by the action of two ubiquitin ligases Ubr1, formerly discovered as recognition component of the N-end rule pathway, and the nuclear ubiquitin ligase San1. For San1-mediated targeting to proteasomal degradation, cytosolic proteins have to be imported into the nucleus. Selection of misfolded substrates for import into the nucleus had remained elusive. This study shows that an increasing molecular mass of substrates prevents nuclear San1-triggered proteasomal degradation but renders them susceptible to cytoplasmic Ubr1-triggered degradation.

Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase.

The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson's disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation.

RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (ß) and Fas2 (α). The (α)6(ß)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the ß-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system.

Mistakes are part of our world and constantly occurring. Due to transcriptional and translational failures, genomic mutations or diverse stress conditions like oxidation or heat misfolded proteins are permanently produced in every compartment of the cell. As misfolded proteins in general lose their native function and tend to aggregate several cellular mechanisms have been evolved dealing with such potentially toxic protein species. Misfolded proteins are mostly recognized by chaperones on the basis of their exposed hydrophobic patches and, if unable to refold them to their native state, are targeted to proteolytic pathways. Most prominent are the ubiquitin-proteasome system and the autophagic vacuolar (lysosomal) system, eliminating misfolded proteins from the cellular environment. A major task of this quality control system is the specific recognition and separation of the misfolded from the correctly folded protein species and the folding intermediates, respectively, which are on the way to the correct folded state but exhibit properties of misfolded proteins. In this review we focus on the recognition process and subsequent degradation of misfolded proteins via the ubiquitin-proteasome system in the different cell compartments of eukaryotic cells. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation.

Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.

Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite-induced degradation of gluconeogenic enzymes.

In the yeast Saccharomyces cerevisiae, key regulatory enzymes of gluconeogenesis such as fructose-1,6-bisphosphatase are degraded via the ubiquitin proteasome system when cells are replenished with glucose. Polyubiquitination is carried out by the Gid complex, a multisubunit ubiquitin ligase that consists of seven different Gid (glucose-induced degradation-deficient) proteins. Under gluconeogenic conditions the E3 ligase is composed of six subunits (Gid1/Vid30, Gid2/Rmd5, Gid5/Vid28, Gid7, Gid8, and Gid9/Fyv10). Upon the addition of glucose the regulatory subunit Gid4/Vid24 appears, binds to the Gid complex, and triggers ubiquitination of fructose-1,6-bisphosphatase. All seven proteins are essential for this process; however, nothing is known about the arrangement of the subunits in the complex. Interestingly, each Gid protein possesses several remarkable motifs (e.g. SPRY, LisH, CTLH domains) that may play a role in protein-protein interaction. We, therefore, generated altered versions of individual Gid proteins by deleting or mutating these domains and performed co-immunoprecipitation experiments to analyze the interaction between distinct subunits. Thus, we were able to create an initial model of the topology of this unusual E3 ubiquitin ligase.

Use of CPY and its derivatives to study protein quality control in various cell compartments.

Mutated derivatives of carboxypeptidase yscY (CPY) are potent substrates to study protein quality control and protein degradation in different cell compartments in yeast. Depending on the subcellular compartment of interest, the design of the model substrate used has to be adapted. Here, we describe the derivatives of CPY used in genetic screens based on a sensitive growth test in order to identify new components of the protein quality control systems in different degradation pathways.

The Cdc48 machine in endoplasmic reticulum associated protein degradation.

The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further delivery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1-Npl4 pulls the misfolded protein out and away from the ER membrane and delivers it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The known details of the Cdc48-Ufd1-Npl4 motor complex triggered process are subject of this review article.

Gid9, a second RING finger protein contributes to the ubiquitin ligase activity of the Gid complex required for catabolite degradation.

The two major antagonistic pathways of carbon metabolism in cells, glycolysis and gluconeogenesis, are tightly regulated. In the eukaryotic model organism Saccharomyces cerevisiae the switch from gluconeogenesis to glycolysis is brought about by proteasomal degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase. The ubiquitin ligase responsible for polyubiquitylation of fructose-1,6-bisphosphatase is the Gid complex. This complex consists of seven subunits of which subunit Gid2/Rmd5 contains a RING finger domain providing E3 ligase activity. Here we identify an additional subunit containing a degenerated RING finger, Gid9/Fyv10. This subunit binds to Gid2/Rmd5. A mutation in the degenerated RING finger of Gid9/Fyv10 abolishes polyubiquitylation and degradation of three enzymes specific for gluconeogenesis.

Mnl2, a novel component of the ER associated protein degradation pathway.

In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY(*) in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.

Yos9, a control protein for misfolded glycosylated and non-glycosylated proteins in ERAD.

The endoplasmic reticulum (ER) is responsible for folding and delivery of secretory proteins to their site of action. One major modification proteins undergo in this organelle is N-glycosylation. Proteins that cannot fold properly will be directed to a process known as endoplasmic reticulum associated degradation (ERAD). Processing of N-glycans generates a signal for ERAD. The lectin Yos9 recognizes the N-glycan signal of misfolded proteins and acts as a gatekeeper for the delivery of these substrates to the cytoplasm for degradation. Presence of Yos9 accelerates degradation of the glycosylated model ERAD substrate CPY∗. Here we show that Yos9 has also a control function in degradation of the unglycosylated ERAD substrate CPY∗0000. It decelerates its degradation rate.

Cdc48: a power machine in protein degradation.

Cdc48 is an essential, highly prominent ATP driven machine in eukaryotic cells. Physiological function of Cdc48 has been found in a multitude of cellular processes, for instance cell cycle progression, homotypic membrane fusion, chromatin remodeling, transcriptional and metabolic regulation, and many others. The molecular function of Cdc48 is arguably best understood in endoplasmic reticulum-associated protein degradation by the ubiquitin proteasome system. In this review, we summarize the general characteristics of Cdc48/p97 and the most recent results on the molecular function of Cdc48 in some of the above processes, which were found to finally end in proteolysis-connected pathways, either involving the proteasome or autophagocytosis-mediated lysosomal degradation.

Dfm1 forms distinct complexes with Cdc48 and the ER ubiquitin ligases and is required for ERAD.

Proteins imported into the endoplasmic reticulum (ER) are scanned for their folding status. Those that do not reach their native conformation are degraded via the ubiquitin-proteasome system. This process is called ER-associated degradation (ERAD). Der1 is known to be one of the components required for efficient degradation of soluble ERAD substrates like CPY(*) (mutated carboxypeptidase yscY). A homologue of Der1 exists, named Dfm1. No function of Dfm1 has been discovered, although a C-terminally hemagglutinin (HA)(3)-tagged Dfm1 protein has been shown to interact with the ERAD machinery. In our studies, we found Dfm1-HA(3) to be an ERAD substrate and therefore not suitable for functional studies of Dfm1 in ERAD. We found cellular, non-tagged Dfm1 to be a stable protein. We identified Dfm1 to be part of complexes which contain the ERAD-L ligase Hrd1/Der3 and Der1 as well as the ERAD-C ligase Doa10. In addition, ERAD of Ste6(*)-HA(3) was strongly dependent on Dfm1. Interestingly, Dfm1 forms a complex with the AAA-ATPase Cdc48 in a strain lacking the Cdc48 membrane-recruiting component Ubx2. This complex does not contain the ubiquitin ligases Hrd1/Der3 and Doa10. The existence of such a complex might point to an additional function of Dfm1 independent from ERAD.