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 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 polyubiqui...