title: Placing a disrupted degradation motif at the C terminus of proteasome substrates attenuates degradation without impairing ubiquitylation.
abstract: Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects.
authors.affiliation: Department of Biological Chemistry, the A. Silberman Institute of Life Sciences, the Hebrew University of Jerusalem, Jerusalem 91904 Israel.
authors.forename: Omri S
authors.initials: OS
authors.lastname: Alfassy
authors.forename: Itamar
authors.initials: I
authors.lastname: Cohen
authors.forename: Yuval
authors.initials: Y
authors.lastname: Reiss
authors.forename: Boaz
authors.initials: B
authors.lastname: Tirosh
authors.forename: Tommer
authors.initials: T
authors.lastname: Ravid
mesh_terms: D020816:Amino Acid Motifs; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018386:Kinetochores / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: C081071:CBF2 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
keywords: Degradation Initiation Site; Endoplasmic Reticulum-associated Degradation; Proteasome; Protein Degradation; Ubiquitin; Ubiquitin Ligase