Proteasome substrate degradation requires association plus extended peptide

Abstract

To determine the minimum requirements for substrate recognition and processing by proteasomes, the functional elements of a ubiquitin-independent degradation tag were dissected. The 37-residue C-terminus of ornithine decarboxylase (cODC) is a native degron, which also functions when appended to diverse proteins. Mutating the cysteine 441 residue within cODC impaired its proteasome association in the context of ornithine decarboxylase and prevented the turnover of GFP-cODC in yeast cells. Degradation of GFP-cODC with C441 mutations was restored by providing an alternate proteasome association element via fusion to the Rpn10 proteasome subunit. However, Rpn10-GFP was stable, unless extended by cODC or other peptides of similar size. In vitro reconstitution experiments confirmed the requirement for both proteasome tethering and a loosely structured region. Therefore, cODC and degradation tags in general must serve two functions: proteasome association and a site, consisting of an extended peptide region, used for initiating insertion into the protease.

Figure 1

Proteasome association is required for degradation. (A) Role of C441 analyzed by competitive inhibition of ODC degradation. The effect on competitive activity of deleting or mutating cODC was analyzed. Here and in subsequent figures, cODC/S and cODC/A designate degradation tags with C441 Ser or Ala mutations. Data are normalized to ODC degradation observed in the absence of competing inhibitor protein and are expressed as percent residual degradation. Incubations with purified rat 26S proteasomes were for 1 h and contained various concentrations of inhibitors, as indicated. The extent of ODC degradation in the absence of inhibitors was 12–13%. Each plot represents the mean of two experiments; error bars indicate standard deviation. (B) Extract was prepared from wild-type cells expressing the indicated proteins. Western blots were developed with anti-GFP. (C) Yeast cells were grown to late logarithmic phase, serially diluted 10-fold and spotted on a non-selective YPD plate or on a plate selective for canavanine resistance. (D) as in (B), but with rpn10Δ cells. (E, F) Pulse–chase analysis of indicated fusion proteins was performed in wild-type (E) or rpn10Δ (F) cells. (G) The data of (F) were scanned and quantitated.

Figure 2

Rpn10-GFP fusion proteins are incorporated into the 26S proteasome. (A) Recovery of Rpn10-GFP with proteasomes and competition by coexpressed Flag-Rpn10. The PRE1-protein A, rpn10Δ cells expressed Rpn10-GFP and, additionally, either Flag-Rpn10 or none. 26S proteasomes were affinity purified, followed by Western blotting with antiserum to GFP, Flag, Rpt5 and the yeast 20S proteasome. Blots of extracts used for affinity purification are shown as well. Arrowheads indicate Pre1-protein A. (B) In vitro competition for proteasome association of Rpn10-GFP. Matrix-bound 26S rpn10Δ proteasomes and ³⁵S-labeled Rpn10-GFP were co-incubated with 10 μg/ml of either BSA or unlabeled Rpn10-GFP, the matrix was washed and the bound proteins subjected to SDS–PAGE and autoradiography. The arrow indicates ³⁵S-Rpn10-GFP, and the asterisk indicates nonspecific band. The input lane contained 1/10 of ³⁵S-Rpn10-GFP used for incubations. (C) In vivo competition for proteasome degradation of Rpn10-GFP proteins. The indicated Rpn10-GFP fusion proteins were expressed in rpn10Δ cells either with or without excess Flag-Rpn10. Crude lysate was prepared from cells and subjected to Western blot analysis with anti-GFP and anti-Flag antibodies, and Anti-Rpt5 as is a loading control.

Figure 3

Both proteasome association and GFP extension are required for in vitro degradation. (A) The indicated Rpn10-GFP fusion proteins were incubated with rpn10Δ proteasomes and degradation measured by Western blotting with anti-GFP. (B) The degradation of Rpn10-GFP-cODC(C441A) protein by the rpn10Δ proteasome was examined as in (A) in the presence of a five-fold molar excess of either GFP-cODC(C441A) or Rpn10. Each plot represents the mean of two (A) or four (B) experiments; error bars (one sided) indicate standard deviation.

Figure 4

Various C-terminal extensions support degradation of Rpn10-GFP fusion proteins. (A) Rpn10-GFP proteins with the indicated C-terminal extensions were expressed and their steady-state level was analyzed by Western blot with anti-GFP. Loading was assessed with anti-Rpt5. (B) Pulse–chase analysis of proteins in (A). (C) Western blot and (D) pulse–chase analysis of Rpn10-GFP fusion proteins with deletions or a duplication of cODC. (E) Quantitation of the data of (D). For (A–D), Western blot and pulse–chase analysis utilized rpn10Δ cells.

Figure 5

Internal sequence can also initiate degradation. (A) Rpn10-GFP proteins of the indicated structure were expressed and their steady-state level was analyzed by Western blot with anti-GFP and anti-Rpt5. (B) Pulse–chase analysis of proteins in (A). (C) Quantitation of the data of (B). For (A–C), rpn10Δ cells were used.