Mutational Analysis of Ubiquitin Shuttle Receptor Docking Sites on the 26S Proteasome
<p>Protein degradation is essential for many basic cellular functions. Most intracellular protein degradation occurs via the ubiquitin proteasome system. Cellular proteins are marked for degradation by the appendage of an ubiquitin chain. Ubiquitin receptor proteins recognize the ubiquitin c...
| Summary: | <p>Protein degradation is essential for many basic cellular functions. Most intracellular protein degradation occurs via the ubiquitin proteasome system. Cellular proteins are marked for degradation by the appendage of an ubiquitin chain. Ubiquitin receptor proteins recognize the ubiquitin chains and play a "garbage man" function in ensuring delivery of the protein trash to the cell’s degradation machinery, the proteasome.</p>
<p>One such class of ubiquitin receptor proteins, known as UBA-UBL proteins, recognizes ubiquitylated substrates and shuttles them to the proteasome. These shuttle receptors include Rad23, Dsk2, and Ddi1. The goal of this dissertation research has been to understand how these UBA-UBL proteins interact with the proteasome. In budding yeast, Saccharomyces cerevisiae, Rpn1 has been proposed to be the major docking site for UBL-containing proteins. More recent studies suggested that proteasome subunits Rpn10 and Rpn13, may also bind UBA-UBL proteins. However, no cis proteasome mutants existed to address these plausible redundant modes of delivery to the proteasome.</p>
<p>The specific aims of this proposal were to: identify the sites on the proteasome that are necessary for specific UBA-UBL receptor docking, to study the consequence of the deletion of these sites (such as the effects on protein turnover), and to assess if the elimination of these sites is the same as elimination of the receptor proteins themselves.</p>
<p>Here, I describe a two-pronged genetic screen I conducted to identify a specific docking site within Rpn1 for UBA-UBL proteins. I uncover a highly conserved residue, D517A that appears to impinge on the ability for both Ddi1 and–in the absence of Rpn13 or the dual absence of the ubiquitin interaction motifs of Rpn10 and Rpn13¬–Dsk2 to interact with the proteasome. However, under no set of genetic conditions does a mutation at Rpn1-D517 have any effect on Rad23 or the UBL-containing ubiquitin isopeptidase Ubp6. Taken together, my observations point to unanticipated diversity and complexity in the mechanisms underlying the recruitment of UBA-UBL proteins to the proteasome.</p>
<p>Hence, I show that docking sites on the proteasome are not completely exclusive, both Ddi1 and Dsk2 share the D517 residue of Rpn1. However, there may be exclusive sites for docking Rad23 and Ubp6. There are also appears to be a layer of complexity and redundancy in docking UBA-UBL proteins to the proteasome. Follow-up studies on these proteasome cis mutatnts have also uncovered roles of the proteasome in regulating mitochondrial protein import and the methyl cycle.</p>
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