title: Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1.
abstract: BACKGROUND
  The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.
  
  
  RESULTS
  To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.
  
  
  CONCLUSIONS
  These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.
authors.affiliation: Division of Biology, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Tara A
authors.initials: TA
authors.lastname: Gomez
authors.forename: Natalie
authors.initials: N
authors.lastname: Kolawa
authors.forename: Marvin
authors.initials: M
authors.lastname: Gee
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D005820:Genetic Testing; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; C108125:DDI1 protein, S cerevisiae; D004268:DNA-Binding Proteins; C100415:DSK2 protein, S cerevisiae; D044783:F-Box Proteins; C490846:RAD23 protein, S cerevisiae; C499663:RPN1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C509156:UFO1 protein, S cerevisiae; D025801:Ubiquitin; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex