RPN4 is required for normal expression of proteasomal subunits. (A and B) Immunoblot analysis of RPN1 (A) and PRE6 (B) that were C-terminally tagged with flag epitope and expressed from their native promoters and a low-copy plasmid in a rpn4Δ S. cerevisiae strain (lane 2) and its congenic wild-type counterpart JD52 (lane 3). Lane 1 in A and B, cells transformed with empty vector. (C) Enhanced expression of RPN4 marginally elevates the level of RPN1. rpn4Δ cells were cotransformed with low-copy plasmids expressing, respectively, RPN1-flag and RPN4 from the P_CUP1 promoter. Increasing concentrations of CuSO₄ were used to induce the expression of RPN4 (lanes 3–7). Lanes 1 and 2 in C, rpn4Δ cells transformed, respectively, with empty RPN1-flag vector and empty RPN4 vector. (D) Immunoblot analyses of C-terminally (RPN4-flag) or N-terminally (ha-RPN4) tagged RPN4 that was expressed from the induced P_CUP1 promoter and low-copy plasmid either in wild-type (RPN4) strain JD52 or in AVY302, a congenic rpn2Δ mutant (see legend to Fig. for details). (E) RPN4 interacts with RPN2 in GST-pulldown assays. Extracts of E. coli expressing RPN4-flag were incubated with glutathione-agarose beads preloaded with the indicated GST fusions or GST alone. The retained proteins were eluted, fractionated, and immunoblotted with anti-FLAG antibody. Approximately equal amounts of different GST fusions were immobilized on glutathione-agarose beads in these assays, as verified by Coomassie staining (data not shown). SDS/PAGE in 6, 8, and 12% gels was used, respectively, in A and C, in D and E, and in B. The asterisk in A, C, and D indicates a crossreacting band.

RPN4 is a short-lived protein degraded by a proteasome-dependent pathway. (A–C) Pulse–chase analysis of C-terminally tagged RPN4 (RPN4-flag) that was expressed from the induced P_CUP1 promoter and low-copy plasmid either in wild-type (RPN4) strain JD52 (A), in a congenic rpn2Δ mutant (B), or in a cim5–1 mutant (C). Cells were labeled at 28°C and chased at 28°C in A and B, and at 37°C in C. The kinetics of RPN4 degradation in JD52 were similar at 28 and 37°C (data not shown). Arrowheads indicate the band of RPN4-flag. (D) Quantitation of the patterns in A–C, by using PhosphorImager (Molecular Dynamics) ●, wild-type cells. ○, rpn2Δ cells. ▵, cim5–1 cells. (E) Pulse–chase analyses of Arg-βgal (R-βgal), derived from Ub-Arg-βgal (, ), and of RPN4-flag (expressed as in A) in uba1–2 () and wild-type cells. Quantitation: ● and ○, Arg-βgal in wild-type and uba1–2 cells, respectively. ▴ and ▵, RPN4-flag in the same strains. (F) The same test proteins in wild-type cells and cells overexpressing Ub^{K48R, G76A}, with quantitation on the right; same designations. The rpn2Δ locus, in the strain AVY302 (see Materials and Methods), was a disruption allele. It was produced through the integration of URA3 at the HindIII site of RPN2 (codon 146) and was identical to the rpn2∷URA3 allele described by Yokota et al. (). The phenotypes of AVY302 and the previously described strain () were similar as well (data not shown). Two other studies reported that rpn2Δ cells were inviable (, ). The disruption of RPN2 in these works was carried out with either TRP1 or ADE2, by using the RPN2 BglII site at codon 38. It remains to be determined whether different integration sites account for different phenotypes described for rpn2Δ strains.

The N-terminal region of RPN4 contains a portable degradation signal. (A) Diagrams of full-length and truncated RPN4. Two putative transcription activation domains are at positions 211–229 and 300–315 (hatched boxes). A putative bipartite nuclear localization signal is at position 381–399 (black box). A putative C₂H₂ finger (residues 477–507) is also indicated. (B) N-terminally truncated RPN4 derivatives are long-lived. RPN4 and its truncated derivatives were expressed from the P_CUP1 promoter and low-copy vector in the JD52 (RPN4) strain. Relative steady-state levels of truncated RPN4 proteins (all of them tagged C-terminally with flag epitope) were determined by immunoblotting, by using SDS/12% PAGE and anti-flag antibody. The bands of proteins with expected sizes are indicated by arrowheads. (C) RPN4_1–151-flag is short-lived. Pulse–chase assays, by using SDS/12% PAGE, were carried out in wild-type (RPN4) cells with RPN4_206–531-flag and RPN4_1–151-flag (indicated by arrowheads) essentially as described in Fig. , except that they were performed at 30°C instead of 28°C. (D) The 151-residue N-terminal fragment of RPN4 contains a portable degron. Pulse–chase assay with RPN4_1–151-βgal fusion (see Materials and Methods) expressed from the P_CUP1 promoter and low-copy vector in the JD52 (RPN4) strain, by using SDS/6% PAGE and immunoprecipitation with anti-βgal antibody.

Cell cycle progression is delayed after G₁ phase in rpn4Δ mutant. (A) A larger fraction of rpn4Δ cells have 2N DNA content in unsynchronized cultures. DNA flow cytometry was carried out with wild-type (RPN4) and rpn4Δ cells. The abscissa ordinate indicate, respectively, the number of cells and relative DNA content. (B and C) Cell cycle progression of rpn4Δ cells is delayed after G₁ phase. Wild-type and congenic rpn4Δ cells were synchronized in late G₁ by using α factor. Initial samples were taken after 2 h of G₁ arrest, and subsequent samples were taken at 20-min intervals after release from the arrest. The samples were analyzed by using DNA flow cytometry (B) and by determining the fraction of budded cells (C). Data shown in C are the means of triplicate measurements.