Figure 4: The stability of Rad23 in vivo is also determined by the presence of an initiation region.
From: Rad23 escapes degradation because it lacks a proteasome initiation region
Rad23 with an N-terminal Flag tag expressed in S. cerevisiae accumulated and protein levels were not affected by MG132 as determined by western blotting for the Flag tag in yeast extracts. Addition of a 39 amino-acids long tail to the C terminus of Rad23 (Rad23-39) or insertion of a 190 amino-acid long linker into loop 3 (Rad23-190
L3) destabilized the protein and reduced steady-state levels in the cell. Inhibition of proteasomal degradation with MG132 stabilized the Rad23 variants and increased their steady-state levels. Rad23 with a 95 amino-acid long tail (Rad23-95) was barely detectable even when the proteasome was inhibited with MG132, but deletion of the UbL domain stabilized the protein sufficiently to allow it to accumulate (not shown). Insertion of a 95 amino-acid linker into loop 2 did not destabilize the protein noticeably (Rad23-95L2). No Flag-tagged Rad23 was detected in lysate of cells transformed with plasmid lacking the Rad23 insert (data not shown) and phosphoglycerate kinase (PGK) was measured as a loading control to allow comparison of protein levels in the different lysates.
