Localization to the Proteasome Is Sufficient for Degradation

Daniel M. Janse, Bernat Crosas, Daniel Finley, George M. Church
2004 Journal of Biological Chemistry  
The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits
more » ... teasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis. ATP-dependent protease complexes degrade the majority of unstable cellular proteins, a process that is conserved across all three kingdoms of life. These molecular machines function both generally in protein turnover and specifically in the regulation of processes such as transcription, apoptosis, antigen presentation, and cell cycle progression (1). A high degree of conservation is evident among them; the archaebacterial and eukaryotic 20S proteolytic core particles share both sequence and structural homology (2), whereas eubacteria have functionally related complexes: ClpYQ, ClpXP, and ClpAP (3-5). The 20S core particle is composed of four stacked heptameric rings structured in an ␣-␤-␤-␣ configuration. Access to the proteolytic central chamber is obstructed at both ends of the cylindrical assembly by N-terminal projections of the ␣-subunits, thus preventing uncontrolled proteolytic degradation (5, 6). In eukaryotes, docking with the 19S regulatory particle (RP) 1 to form the complete 26S proteasome is sufficient to relieve this block, opening a channel into the core (6, 7). Eukaryotes have evolved an elaborate system that operates in conjunction with the proteasome to facilitate the temporal and specific regulation of intracellular proteolysis. Most proteins are targeted for degradation through ubiquitination, mediated by the E1, E2, E3 ubiquitin (Ub) conjugation machinery. These three consecutively acting enzymes are necessary for target recognition, transfer of a ubiquitin moiety to the substrate, and subsequent elongation of the ubiquitin branched chain (8). Modularity and the large number of E2 Ub-conjugating enzymes and E3 Ub ligases allow for greater specificity and flexibility in recognizing a diverse range of substrates. Once a protein is polyubiquitinated, it is targeted to and degraded by the 26S proteasome. The polyubiquitin chain is thought to play two possible roles. The first is to target the protein to the proteasome; the second is to initiate the process of degradation. The targeting hypothesis is supported by the identification of several proteasome subunits that either bind or crosslink to ubiquitin chains (9, 10). Hypotheses for how ubiquitin-dependent initiation of degradation might occur include allosteric regulation, channel opening, and assistance in the unfolding of the target (11). However, little data have been reported to support these ideas. The elucidation of the mechanism for proteolysis of ornithine decarboxylase (ODC) established that polyubiquitination is not necessary for proteasome-mediated degradation (12). ODC is an enzyme whose degradation is mediated by its binding to the cofactor antizyme 1 (AZ1). Once bound, ODC-AZ1 can be recognized by the proteasome, and ODC is degraded in an ubiquitin-independent manner. However, it is unclear whether the means by which AZ1 promotes degradation differs fundamentally from that of polyubiquitin chains. A recent study demonstrated that ODC-AZ1 competes with substrate-linked and free polyubiquitin chains for the occupancy of the same binding site on the proteasome (13). Thus, the mechanism for the degradation of ODC seems to represent a specialized evolutionary adaptation that closely mimics ubiquitination. The binding of ubiquitin-conjugated substrates (or ODC-AZ1) to the proteasome may itself serve as the activation step in proteolytic degradation. We hypothesize that such an activation step is not necessary, that localization to the proteasome can be sufficient for degradation. In this study, we support this hypothesis by demonstrating proteasome-mediated degradation both in vivo and in a purified in vitro system, while artificially bypassing the need for ubiquitin-dependence. EXPERIMENTAL PROCEDURES Construction of Parental Strain DY001-All experiments were performed in derivatives of strain DY001 to ensure that the components of the heterodimerization system would minimally interact with endogenous proteins, thus preventing cell cycle arrest and mislocalization of the reporter upon the addition of rapamycin (14). The Fpr1-rapamycin binding domain (nucleotides 5656 -6243) of the dominant allele TOR1-2 was amplified from strain JHY17-9C (15) and subcloned into the integrating plasmid pRS306 (16). This vector was then digested with HindIII to cut once within TOR1-2 and transformed into the strain BY4742 ⌬fpr1::kan r (Research Genetics). Integration and subsequent loop-out was selected for on the appropriate plates. The correct strain was verified by PCR and sequencing. Downloaded from Genomic Tagging of Proteasome Subunits-Tagging of proteasome subunits was performed both by homologous recombination of linear fragments containing 40 bp of flanking homology to the target site and by two-step integration with a non-replicating plasmid. FPR1 was amplified by PCR from the strain FY4 (17) and subcloned into the plasmid pUG-spHIS5 (18) with a C-terminal hemagglutinin (HA)-tag, forming FPR1-HA-pUG-spHIS5. Integration primer pairs were designed for tagging each of four proteasome subunits (PRE10, RPN2, RPN6, RPN11). For each pair, one primer contained 40 bp of genomic homology to the 3Ј end of the proteasome subunit, excluding the stop codon, and 20 bp of homology to the 5Ј end of FPR1 on pUG-spHIS5, excluding ATG. The second primer contained 40 bp of genomic homology ϳ50 bp downstream of the proteasome subunit gene stop codon and 20 bp of homology to pUG-spHIS5 immediately downstream of the spHIS5 marker flanked by loxP sites. Two confirmatory primers were also designed that flanked the integration site of each proteasome subunit. Strain DY001 was transformed with pSH47, a plasmid with a galactose-inducible cre gene and a URA3 selection marker (19). A 2-kb linear fragment from FPR1-HA-pUG-spHIS5 was amplified using each integration primer pair to generate linear 2-kb fragments suitable for genomic integration. 15 g of each fragment was transformed into DY001 carrying pSH47, and selection was performed on SC-URA-HIS. Colonies were then picked and streaked onto SC-URA GAL to induce cre and to select for the loopout of the spHIS5 marker. Colonies were finally streaked onto a 5-fluoroorotic acid-containing plate to remove pSH47. All tagged subunits were verified by sequencing. Approximately 400 -500 bp of the carboxyl-terminal end (without the stop codon) and 3Ј untranslated region of proteasome subunits RPT2, RPT5, and RPN10 were amplified from strain FY4. Each pair was subcloned into the integration plasmid pRS306 (16) along with FPR1 so that the final structure at the cloning site was 5Ј-proteasome subunit C-term-FPR1-proteasome subunit UTR. Each derivative of pRS306 was cut at a unique site within the carboxy terminus of the proteasome subunit and transformed into DY001. Selection for integration was done on SC-URA; loopout of the marker was on 5-fluoroorotic acidcontaining plates. All tagged subunits were verified by sequencing. ⌬pdr5 strains were generated by recombination of a URA3 marker flanked by 40 bp homologous to sequence immediately 5Ј and 3Ј to genomic PDR5
doi:10.1074/jbc.m402954200 pmid:15039430 fatcat:2sl5tzpslvdvjbp2vt6zzcgwka