Figure 3.

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. 2, 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.