Abstract
Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome. In this study, we isolated another PCI proteasome subunit, Rpn7, as a high copy suppressor that rescued the proteasome defects in yin6 null cells. To better define the function of the PCI domain, we aligned protein sequences to identify a conserved leucine residue that is present in nearly all known PCI domains. Replacing it with aspartate in yeast Rpn7, Yin6, and Rpn5 inactivated these proteins, and mutant human Int6 mislocalized in HeLa cells. Rpn7 and Rpn5 bind Rpn9 with high affinity, but their mutant versions do not. Our data suggest that this leucine may interact with several hydrophobic amino acid residues to influence the spatial arrangement either within the N-terminal tandem alpha-helical repeats or between these repeats and the more C-terminal winged helix subdomain. Disruption of such an arrangement in the PCI domain may substantially inactivate many PCI proteins and block their binding to other proteins.
Highlights
The 26 S proteasome contains a 20 S catalytic core, which has a cylindrical shape and on its own is capable of degrading small peptides [1]
In a previous bioinformatic analysis of proteasome-COP9-initiation factor (PCI) architecture [6], we predicted that the N-terminal subdomain of typical PCI domains most likely resembles the tetratricopeptide repeat (TPR), which consists of pairs of ␣-helices that are oriented in an antiparallel manner
We found that overexpressing Rpn7 rescued canavanine hypersensitivity of yin6⌬ cells (Fig. 1B)
Summary
Strains and Growth Conditions—The parental wild-type S. pombe strain used in this paper is SP870 (h90, ade6-M210, leu132, ura4-D18). Site-directed mutagenesis was carried out to mutate the rpn coding sequence in pBSRPN5-HAKanR [14] to create pBSR5LD-HAKanR This plasmid was digested by XhoI and SmaI to release a fragment containing rpn, and this fragment was used to transform a rpn5b⌬ strain (RPN5bA) [14]. These data indicate that the ts growth defect is caused amounts of proteins were analyzed by immunoblotting with an by loss of Rpn function. We used this strain, called ubiquitin antibody (1:100; Sigma)
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