Proteasome substrates and N end rule degron-mediated degradation. (a) Structure of barnase bound to its inhibitor barstar [PDB ID 1BRS] and the molecular surface of this complex shown in cartoon representation using PyMol (DeLano Scientific LLC, Palo Alto, CA; www.pymol.org). (b) Barstar and barnase hybrid proteins. Barstar was targeted to the proteasome in reticulocyte lysate by an N end rule tag, which was composed of an N-terminal ubiquitin followed by an unstructured extension containing two lysine residues (N-degron). The N-degron was attached to the N terminus of barstar through an intervening DHFR domain. Barnase was targeted to the proteasome through an N end-rule degron in which the linker contained an additional 15 amino acids. Other barnase constructs contained N-terminal unstructured initiation sites derived from cytochrome b₂ or lac repressor. (c) Proteolysis of barstar by the ubiquitin proteasome system in rabbit reticulocyte lysate. Incubation of N-degron-DHFR-barstar in reticulocyte lysate leads to its rapid degradation, which was inhibited in the absence of ATP, by addition of an excess of a ubiquitin mutant that inhibits poly-ubiquitination because it lacks all Lys residues, and by the proteasome inhibitor MG132 (1). Vertical lines separate radiogram images from different gels that were acquired simultaneously and set to the same exposure. Ubiquitination of substrates occurs at the lysine residues in the linker region of the N-degron, and deletion of the linker region in N-degron eliminates substrate ubiquitination as well as its degradation.

The proteasome degrades an unubiquitinated subunit in a heterodimeric complex. Radiolabeled barnase protein bound to its ligand barstar or to barstar containing the N-degron was degraded by the proteasome at 30 °C. (a) Barnase containing an N-degron and an initiation site was efficiently degraded by the proteasome when in complex with its ligand barstar. The N-degron is modified with many ubiquitin moieties, but, for simplicity, the polyubiquitin modification is indicated by four ubiquitin moieties in the schematic representation. (b) Barnase bound to N-degron-DHFR-barstar was not degraded by the proteasome unless (c) initiation sites derived from lac repressor (open circles) or cytochrome b₂ (filled circles) were attached to barnase's N terminus. (d) Barnase proteins containing N-terminal initiation sites were not degraded when bound to barstar was not ubiquitinated because it lacked the N-degron. (e) Proteolysis of barnase bound to N-degron-DHFR-barstar adaptor was dependent on an active ubiquitin-proteasome system and was inhibited in the absence of ATP (filled squares) by the proteasome inhibitor MG132 (1) (open circles) and by the addition of excess of a ubiquitin mutant that inhibits poly-ubiquitination because it lacks all Lys residues (open squares). (f) Size exclusion chromatography elution profiles of [³⁵S]-labeled barnase containing an unstructured initiation site bound to barstar or a barstar hybrid containing an N-degron. (g) Barnase containing an initiation site was assembled with N-degron-DHFR-barstar in the absence (filled circles) or presence (open squares) of 10-fold excess of untagged barstar and was degraded by the proteasome in rabbit reticulocyte lysate at 30 °C. Graphs show one representative dataset of at least three independent experiments.

trans initiation by the proteasome. (a) Coomassie blue stained SDS-PAGE gels separating 15 μg of purified yeast 26S proteasome. Proteasome subunits are labeled following published reports49. (b) Barstar was targeted to the proteasome by an N-terminal Ub₄ degron composed of four ubiquitin moieties fused in frame and then through a 16-residue linker to barstar. Barnase contained a 35 amino acid N-terminal unstructured region derived from cytochrome b₂ (USR1-barnase). (c) Coomassie blue stained SDS-PAGE gels separating 1 μg of indicated proteins. (d) 0.5 μM USR1-barnase was assembled with 1 μM (2X) or 0.1 μM (0.2X) barstar (open symbols) or Ub₄-barstar (filled symbols), and the complex was degraded by purified yeast proteasome. The graph shows the amount of barnase remaining (quantified by Western blotting) as a function of reaction time. (e) 0.5 μM USR1-barnase was assembled with Ub₄-barstar (0.2 μM; filled squares) and degraded. A 10-fold excess of untagged barstar (open symbols) was added at the beginning of (squares) or 20 min after starting the reaction (circles), as indicated by bold arrows. (f) Degradation required an unstructured initiation site on the barnase substrate. The proteasome did not degrade barnase that lacked an unstructured region even when bound to 1 μM (2X) or 0.1 μM (0.2X) Ub₄-barstar. (g) Schematic representation of trans initiation. A ubiquitinated protein may act as a recycling adaptor to shuttle its binding partner to the proteasome. The sketch shows a proteasome particle capped by two PA700 regulatory particles but other forms also exist. Graphs show one representative dataset of at two (e) or three independent experiments.

Substrate selection in reticulocyte lysate. (a-d) [³⁵S]-methionine labeled barnase containing an initiation site was bound to an excess of N-degron-DHFR-barstar (0.25 μM) and degraded by the proteasome in reticulocyte lysate at 30 °C in the absence (a, b) or presence (c, d) of 100 μM methotrexate (2). Lanes in the autoradiograms contain equal samples of the reaction at the indicated times. (a, c) Trace amounts of radiolabeled N-degron-DHFR-barstar were added to reactions to visualize its stability in the reaction by SDS-PAGE and autoradiography. (b, d) Degradation of the barnase substrate was followed in parallel by SDS-PAGE and autoradiography. (e, f) Quantifications of the full-length protein (indicated by arrows) and its ubiquitinated forms (indicated by square brackets) in the autoradiograms are plotted as the amounts of protein remaining as a percentage of the total protein at the beginning of the reaction. Both rapidly degrading and methotrexate (2)-stabilized barstar subunit (e) functioned similarly in targeting the associated barnase for degradation (f). Graphs show one representative dataset of five independent experiments.

Substrate selection by the purified proteasome. (a-c) 0.5 μM barnase containing a N-terminal initiation site (red square) was bound to 0.05 μM Ub₄-barstar (a, blue circles) or 0.05 μM Ub₄-USR2-barstar (b, blue circles) or 0.05 μM Ub₄-DHFR-barstar (c, blue circles) and the protein complex was degraded by purified proteasome at 30 °C and analyzed by Western blotting (top). Quantifications of the gels are shown as graphs plotting the amount of barnase (red) and barstar (blue) protein remaining at different reaction times as a percentage of protein amounts at the beginning of the reaction. Note that the barnase substrate is present in 10 fold molar excess over the barstar adaptor. Graphs show one representative dataset of two (b) or three independent experiments.

Subunit-specific degradation by the proteasome. (a-d) 0.5 μM Barnase containing a N-terminal initiation site (red squares) was bound to 0.5 μM Ub₄-barstar (a, blue circles) or 0.5 μM Ub₄-DHFR-barstar (b, blue circles) or 0.5 μM Ub₄-USR3(Ser-rich)-barstar (c, blue circles) or 0.5 μM Ub₄-USR4-barstar (d, blue circles) and degraded by purified proteasome at 30 °C. Reactions were separated by SDS PAGE and analyzed by Western blotting. The graphs plot the amount of barnase substrate (red) and barstar adaptor (blue) protein remaining at different reaction times as a percentage of protein amounts at the beginning of the reaction. Note that the barnase substrate is present in equimolar concentration to the barstar adaptor. (e) Schematic representation of the substrate selection by the proteasome. Here, both subunits in the complex are accessible to the proteasome and the protease selects the subunit with the preferred initiation site for degradation (path 1 and 2). If more than one subunit contains a proteasome initiation site, multiple subunits might be degraded simultaneously (path 3) or the proteasome might switch stochastically between pathways 1 and 2. The sketch shows a proteasome particle capped by two PA700 regulatory particles. Proteasome particles may contain only one PA700 regulator or they may be activated by complexes that do not bind ubiquitin. Graphs show averages of two (c) or three independent experiments.