Not4 is important for ubiquitin homeostasis in the cell. (A) Serial dilutions of the indicated mutants, containing a NOT4-URA3 plasmid and either a vector alone or a LEU2 plasmid expressing Not4, were spotted on plates with 5-fluoroorotic acid (FOA) and left to grow for several days. (B) Exponentially growing wild-type (WT) and not4Δ cells were collected at an OD₆₀₀ of 1.5. Total protein extracts were prepared as described previously (30), and proteins were separated on a 4 to 12% gel (for polyubiquitinated proteins [poly Ub]) or on a 17% gel (for free ubiquitin [free Ub]), followed by immunoblotting with antiubiquitin antibodies or anti-Egd2 antibodies as a loading control. Numbers at the left are molecular masses (in kilodaltons). (C) The indicated strains were grown exponentially, and serial dilutions were spotted on yeast extract-peptone-dextrose (YPD).

Proteasome integrity is altered in not4Δ cells. (A) Total cellular extracts were prepared from wild-type (MY1) or not4Δ mutant (MY3595) cells collected at an OD₆₀₀ of 3.0. One-hundred-microgram samples of total protein extracts (TE) were separated on a 3.5% native gel and analyzed for proteasome activity with Suc-LLVY-AMC without and with SDS. RP₂-CP and RP₁-CP are double- and single-capped proteasomes. Fifty-microgram samples of the same extracts were loaded onto SDS-PAGE gels and immunoblotted with anti-Rpt1 antibodies as a loading control. (B) Twenty-microgram samples of 26S holoenzymes, purified from wild-type (sDL133) or not4Δ mutant (MY6467) cells were separated on a 3.5% native gel and either stained with Coomassie blue, tested for activity, or analyzed for the presence of Rpt1 by immunoblotting. The same samples were loaded onto SDS-PAGE gels and immunoblotted with anti-Rpt1 antibodies as a loading control (bottom).

RP species from the not4Δ mutant that are not incorporated into salt-resistant RP-CP complexes are unstable. Total extracts from wild-type or not4Δ cells expressing Rpn11-ProtA were split into four portions. The NaCl concentration in each sample was adjusted to allow purification of holoenzyme (0.1 M), RP (0.5 M), or lid (0.75 and 1 M) (33). (A) Purified complexes were analyzed on native gel and either tested for activity or immunoblotted with antibodies against Rpn12, Rpt1, and α1–7. (B) The same purified complexes were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies.

Not4 genetically, physically, and functionally interacts with Ecm29. (A) The indicated strains were spotted on YPD plates with or without CHX and left to grow at 30 or 37°C. (B) ecm29Δ cells expressing HA-Ecm29 from an episome (the wild type, the not4Δ mutant, and two different transformants of the not4Δ mutant with the Myc₆-Not4 plasmid) were collected at an OD₆₀₀ of 0.8, and total proteins were analyzed by immunoblotting with anti-HA antibodies. (C) Thirty-microgram samples of total protein extracts from not4Δ cells expressing the Myc-Not4 derivatives were analyzed by immunoblotting with antibodies against Ecm29, Myc, and Egd2 as a control for loading. Numbers at the left are molecular masses (in kilodaltons). (D) Exponentially growing ecm29Δ or ecm29Δ not4Δ cells expressing HA-Ecm29 were treated or not treated with CHX for the indicated times. Equivalent amounts of total extract were analyzed by immunoblotting with antibodies against HA or Egd2 as a control for loading. (E) Ubiquitinated proteins were purified from ecm29Δ or ecm29Δ not4Δ cells expressing HA-Ecm29 and His₆-ubiquitin (43). Total extract (TE) or ubiquitinated proteins bound to the Ni-resin (UB) were analyzed by immunoblotting with anti-HA antibodies. (F) Total protein extracts (TE) from cells expressing Myc-Not4 were incubated with antibodies against the Myc tag (IP_Myc) or against Ecm29 (IP_Ecm29). The presence of Ecm29 and Myc-Not4 in the immunoprecipitates was evaluated by immunoblotting. The strains expressing endogenous untagged Not4 (left) and not expressing Ecm29 (right) were used as controls for the specificity of the immunoprecipitation.

Structure-function analysis of Not4. (A) Schematic representation of the Not4 protein, with its ring finger domain (RING), putative coiled-coil domain, and RNA recognition motif (RRM). (B) ubp6Δ not4Δ cells carrying a NOT4-URA3 plasmid were transformed with LEU2 plasmids expressing the indicated Myc-Not4 derivatives and analyzed on FOA plates as described for Fig. 1A. (C) Cycloheximide (CHX) sensitivity of not4Δ cells expressing the Myc-Not4 derivatives. (D) Total protein extracts were prepared from exponentially growing not4Δ cells expressing the Myc-Not4 derivatives and analyzed as described for Fig. 1B. Numbers at the left are molecular masses (in kilodaltons). (E) Not5 was immunoprecipitated (IP_Not5) from the total extracts prepared from the same cells as those used for panel D (TE). Samples were analyzed by 4 to 12% SDS-PAGE and immunoblotting for the presence of Not5 or Myc-Not4.

RP and CP form salt-resistant complexes in not4Δ cells. Total cellular extracts from wild-type or not4Δ cells expressing Rpn11-ProtA (for RP purification) and Pre1-ProtA (for CP purification) were incubated with IgG beads. After the beads were washed with a high-salt buffer, RP and CP were eluted from the column by TEV cleavage (see Materials and Methods) and analyzed by SDS-PAGE (A) or by immunoblotting (B) for the presence of Rpt1 or CP subunits (α7 or α1–7). Numbers to the left are molecular masses (in kilodaltons). The asterisk in panel B marks a nonspecific band. (C) The same samples were analyzed on a 3.5% native gel and either stained with Coomassie blue (left), tested for activity (middle), or analyzed for the presence of Rpt1 and α1–7 by immunoblotting (two right-most blots). (D) Rpn11-ProtA was affinity purified in a high concentration of salt (0.5 M) from the not4Δ mutant expressing the indicated Myc-Not4 derivatives. Purified proteins were then separated on a 3.5% native gel and either tested for activity or transferred to nitrocellulose, probed with Ponceau S, and analyzed for the presence of Rpt1 or α1–7. The same purified proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against Rpt1 or α1–7 (bottom). (E) In vitro reconstitution of proteasomes. Separately purified RP and CP from wild-type and not4Δ cells (the same as those used for panel A) were mixed in the following combinations: wild-type RP (lane 1), wild-type RP plus wild-type CP (lane 2), wild-type RP plus CP_not4Δ (lane 3), RP_not4Δ plus wild-type CP (lane 4), wild-type CP (lane 5), RP_not4Δ plus CP_not4Δ (lane 6), CP_not4Δ (lane 7), and RP_not4Δ (lane 8). Samples were incubated at 30°C for 30 min, loaded onto a 3.5% native gel, and tested for activity or immunoblotted with anti-Rpt1, α1–7, or Rpn5 antibodies.

Not4 associates with RP species present in holoenzyme but not with purified RP. (A) 26S holoenzyme was purified via Rpn11-ProtA from not4Δ cells (MY7820) expressing Myc-Not4. Total extracts (TE) (lane 1) and purified proteins (purif) (lane 2) were analyzed by immunoblotting for the presence of Rpt1, Rpn8, α1–7, and Myc-Not4. (B) Holoenzymes from the wild type were immobilized on IgG beads via Rpn11-ProtA and incubated with separately purified Myc-Not4. After the beads were washed, proteasome was eluted by TEV cleavage. Total extracts (lane 1), added Myc-Not4 (Not4) (lane 2), and proteins in the TEV eluate (lane 3) were analyzed by immunoblotting for the presence of Rpt1, Rpn8, α1–7, and Myc-Not4. (C) Myc-Not4-ProtA was immobilized on IgG beads. Separately purified holoenzyme and RP from cells expressing Rpn5-ProtA were added to the beads. After the beads were washed, Myc-Not4 was eluted by TEV cleavage. Total extracts (lane 1), added holoenzyme (Holo) and RP (lanes 2 and 3, respectively), and the proteins in the TEV eluates (lanes 4 and 5, respectively) were analyzed by immunoblotting for the presence of Rpn8, α1–7, and Myc-Not4.

Some of Not4's impact on the proteasome is through Ecm29. (A) Rpn11-ProtA was purified at 0.5 M NaCl from cells of the wild type (MY5559) (lane 1), 2 different not4Δ mutants (MY7367 [lane 2] and MY7820 [lane 3]), the ecm29Δ mutant (MY7827) (lane 4), the ecm29Δ not4Δ mutant (MY7822) (lane 5), and the ecm29Δ not4Δ mutant expressing Ecm29 from a multicopy plasmid (MY7822 plus pECM29 mc) (lane 6) or from a single-copy plasmid (MY7822 plus pECM29 sc) (lane 7). One hundred micrograms of total extracts used for purification was loaded on a 3 to 12% native gel and analyzed for activity (top) or by immunoblotting with PAP antibodies (right). Purified RPs were loaded on a 3 to 12% native gel and analyzed for activity (middle) or by Coomassie blue staining (bottom). (B) The same samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (C) Ecm29 was affinity purified from wild-type or not4Δ cells. Total extract (TE) and purified proteins (purif) were analyzed by immunoblotting with the indicated antibodies.