title: Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.
abstract: Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones.
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Maurer
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Spear
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Allen T
authors.initials: AT
authors.lastname: Yu
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Saba
authors.initials: S
authors.lastname: Shahzad
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 michaelis@jhmi.edu.
authors.forename: Susan
authors.initials: S
authors.lastname: Michaelis
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation / Q000235:genetics; D017930:Genes, Reporter; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C542288:SSA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; D025801:Ubiquitin; C070319:YDJ1 protein, S cerevisiae; C552998:Ltn1 protein, S cerevisiae; C519403:Rkr1 protein, S cerevisiae; C500396:SAN1 protein, S cerevisiae; C091362:SSM4 protein, S cerevisiae; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae
keywords: E3 ligase Doa10; Ltn1; protein quality control; proteostasis; ubiquitin proteasome system