Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

Abstract

Eukaryotic cells maintain proteostasis by quality control (QC) degradation. These pathways can specifically target a wide variety of distinct misfolded proteins, and so are important for management of cellular stress. Although a number of conserved QC pathways have been described in yeast, the E3 ligases responsible for cytoplasmic QC are unknown. We now show that Ubr1 and San1 mediate chaperone-dependent ubiquitination of numerous misfolded cytoplasmic proteins. This action of Ubr1 is distinct from its role in the "N-end rule." In this capacity, Ubr1 functions to protect cells from proteotoxic stresses. Our phenotypic and biochemical studies of Ubr1 and San1 indicate that two strategies are employed for cytoplasmic QC: chaperone-assisted ubiquitination by Ubr1 and chaperone-dependent delivery to nuclear San1. The broad conservation of Ubr ligases and the relevant chaperones indicates that these mechanisms will be important in understanding both basic and biomedical aspects of cellular proteostasis.chaperone | proteostasis | misfolding P rotein quality control (QC) functions to ensure that damaged and misfolded proteins are maintained at acceptable levels to limit their stress-causing, or proteotoxic, effects. One strategy of protein QC is the selective degradation of misfolded proteins. For degradative QC pathways to be effective, they must be specific for aberrant proteins; sufficiently general to recognize selectively common structural hallmarks shared by numerous unrelated proteins; and physiologically important, better allowing the cell to survive proteotoxic stress. Because protein QC underlies many pressing maladies, such as parkinsonism, cystic fibrosis, and aging, discovery of the rules of substrate selectivity and destruction is a key step in understanding these conditions and designing appropriate therapeutical interventions to combat them.In eukaryotes, the ubiquitin proteasome system is employed in the selective degradation of many proteins (1). A substrate protein is marked for degradation by assembly of a polyubiquitin chain, initiated by covalent addition of the small (7.6 kDa) protein ubiquitin to a lysine in an isopeptide bond, followed by iterative addition of the next ubiquitin to the previously added one to create a polyubiquitin chain that is uniquely recognized by the 26S proteasome. Protein ubiquitination is catalyzed by a three-enzyme cascade. The single E1 ubiquitin-activating enzyme hydrolyzes ATP to acquire ubiquitin in labile thioester linkage, which is then transferred in thioester linkage to one of a small group of E2s or ubiquitin-conjugating enzymes (UBCs). E2-bound ubiquitin is finally transferred to an isopeptide linkage on the target protein or the growing polyubiquitin chain by the action of the E3 ubiquitin ligase. It is the E3 ubiquitin ligase that determines the specificity of a given ubiquitination process; identifying and understanding the E3s involved in a degradative pathway are thus key parts of understanding the mechanisms of substrate selection and modification.E3s ...

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

“…This study adds another layer of complexity to the known mechanisms of ERAD and underscores that previously accepted unidirectional pathways of ubiquitin-triggered degradation of proteins can intersect in a cell: the cytosolic ubiquitin ligase Ubr1 known to be involved in the quality control of cytosolic N-end rule substrates (47,48), as well as misfolded cytoplasmic proteins (25)(26)(27), now can be connected to ERAD (this study). On the other hand, the ERAD ligase Doa10, known mainly to facilitate degradation of ERAD substrates with a misfolded cytosolic domain, also ubiquitinates cytosolic N-acetylated substrates or substrates containing specific C-terminal appendages and targets them for degradation (49)(50)(51).…”

“…Recently, the cytosolic RING (really interesting new gene) -type ubiquitin ligase Ubr1 was linked to ubiquitination and degradation of misfolded proteins in the cytosol (25)(26)(27). Therefore, we considered this ligase to ubiquitinate Ste6* in the absence of the canonical ERAD ligases.…”

Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants

“…Two ubiquitin E3 ligases, San1p and Ubr1p, have been identified as responsible for marking misfolded cytoplasmic proteins for degradation in nuclear proteasomes (37,38). We find that neither Btn2 nor Cur1 overproduction curing depends on San1p or Urb1p (Fig.…”

“…The failure of bmh1Δ to detectably alter the efficiency of prion curing by overproduced Btn2p, Cur1p, or Hsp42p suggests that these proteins are not acting through the yeast aggresome defined by Wang et al (16). Ubr1p and San1p are E3 ubiquitin ligases involved in targeting misfolded cytoplasmic proteins to proteasomes in the nucleus (37,38). The [URE3] prion is certainly a misfolded form of Ure2p that is in the cytoplasm, but it seems that Btn2p and Cur1p are not working through these ubiquitin ligases.…”

The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control

“…PQC is a collection of critical pathways within the ubiquitinproteasome system that are responsible for removing misfolded proteins from the cell (16). PQC systems are prevalent throughout the cell and can be found at important multisubunit complexes such as the ribosome (17)(18)(19)(20) and in the cytoplasm (21)(22)(23)(24)(25) and organelles such as the endoplasmic reticulum (26,27), mitochondria (28), and nucleus (29). Not surprisingly, the breakdown of normal PQC function may lead to several human diseases.…”