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.2006 Apr 19;25(8):1720-9.
doi: 10.1038/sj.emboj.7601058. Epub 2006 Apr 6.

Glycine-alanine repeats impair proper substrate unfolding by the proteasome

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Glycine-alanine repeats impair proper substrate unfolding by the proteasome

Martin A Hoyt et al. EMBO J..

Abstract

Proteasome ATPases unravel folded proteins. Introducing a sequence containing only glycine and alanine residues (GAr) into substrates can impair their digestion. We previously proposed that a GAr interferes with the unfolding capacity of the proteasome, leading to partial degradation of products. Here we tested that idea in several ways. Stabilizing or destabilizing a folded domain within substrate proteins changed GAr-mediated intermediate production in the way predicted by the model. A downstream folded domain determined the sites of terminal proteolysis. The spacing between a GAr and a folded domain was critical for intermediate production. Intermediates containing a GAr did not remain associated with proteasomes, excluding models whereby retained GAr-containing proteins halt further processing. The following model is supported: a GAr positioned within the ATPase ring reduces the efficiency of coupling between nucleotide hydrolysis and work performed on the substrate. If this impairment takes place when unfolding must be initiated, insertion pauses and proteolysis is limited to the portion of the substrate that has already entered the catalytic chamber of the proteasome.

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Figures

Figure 1
Figure 1
Ribbon diagrams of interactions stabilizing β-sheet domains. (A) Mouse ODC (PDB id: 7ODC); inset shows a global view of the monomer. The C-terminus (residues 403–418) is shown in orange and the N-terminus in blue. The side chains of residues involved in stabilizing interactions described in Table I are shown as solid sticks, with carbon atoms in green, oxygen in red and nitrogen in blue. Residues 30–35 are missing from the crystal structure. (B) Human DHFR (PDB id: 1DHF); inset shows a global view of the monomer. The C-terminus is shown in orange and the ‘safety belt' in blue. Stabilizing residues are shown as solid sticks. For N29 and G164, only the interaction backbone moieties are shown.
Figure 2
Figure 2
Mutations that disrupt ODC-stabilizing interactions reduce the accumulation of degradation intermediates. (A) Pulse-chase analysis of yeast cells expressing wild-type or mutant ODC::GAr30 fusion proteins. The position of full-length ODC::GAr30 (solid arrow) or degradation intermediates (dashed arrows) is indicated. (B) Quantitation of ODC::GAr30 turnover data from (A), presented as the mean of three independent experiments. Error bars represent the standard deviation. The values for wild-type ODC::GAr30 (•), Δ17ODC::GAr30 (□) and Δ11ODCL14D::GAr30 (▴) are expressed as a percentage of the total of the full-length parent fusion protein and its degradation intermediates at time zero. The changes in the wild-type and mutant proteins are compared for the parent molecule (middle panel), its degradation intermediates (bottom panel) and the sum total of these (top panel) at each time point. (C) Pulse-chase analysis of ODC::GAr30 fusion proteins containing the C441A mutation in the ODC degradation tag. Arrows indicate bands as in (A).
Figure 3
Figure 3
The accumulation of DHFR-GAr30-C37 degradation intermediates is influenced by the stability of the DHFR domain. (A) Pulse-chase analysis of wild-type and mutant DHFR-GAr30-C37 proteins. (B) Pulse-chase analysis of fusion proteins containing the C441A mutation in the ODC degradation tag. (C) Pulse-chase analysis of DHFR-GAr30-C37 in yeast cells either with (+MTX) or without (−MTX) the addition of 20 μM methotrexate. Arrows indicate bands as in Figure 2.
Figure 4
Figure 4
The positions of cutting sites that generate stable intermediates are not altered by extending the degradation tag. Pulse-chase analysis of yeast cells expressing ODC::GAr30 fusion proteins with wild-type (C37) or extended (C75) degradation tags. The parent bands and common intermediates are indicated by arrows as in Figure 2.
Figure 5
Figure 5
The effects of spacing between GAr and β-sheet domain on production of intermediates. (A) Schematic of ODC::GAr30 constructs containing spacers of varying length between the ODC C-terminal β-sheet and the GAr30 domains. The number of spacer residues is indicated below each construct. (B) Pulse-chase analysis of cells expressing constructs in (A). (C) Quantitation of turnover data for ODC[n]GAr30 fusions in (B) with spacer lengths of 0 (•), 7 (□), 13 (Δ), 19 (▪) or 25 (○) residues were analyzed as in Figure 2B.
Figure 6
Figure 6
Pulse-chase analysis of ODC::GAr30 linker constructs with C-terminal extensions of fixed size. (A) Schematic of ODC::GAr30 constructs in which the context of the Gly–Ala repeat is varied with respect to its flanking linker sequences. The length of each region in residues is indicated below each construct. (B) Pulse-chase analysis of yeast transformants expressing the fusion proteins shown in (A). Arrows indicate bands as in Figure 2.
Figure 7
Figure 7
Degradation intermediates do not cofractionate with proteasomes. Lysates were prepared from cells expressing ODC::GAr30 and bearing either no proteasome affinity tag or a Protein A (ProA) tag associated with either the Pre1 subunit of the 20S core particle or the Rpn11 subunit of the 19S regulatory complex. Following affinity purification, fractions designated total (T), supernatant (S) and matrix-associated (M) were subjected to SDS–PAGE and immunoblotting; M fractions were overloaded 20-fold compared to the T and S fractions to facilitate visualization of affinity-fractionated, proteasome-associated proteins. Immunoblotting was performed with antibodies specific for the FLAG epitope in the ODC::GAr30 substrate (solid arrow) and intermediates (dashed arrow), Rpt5, 20S or Sem1 proteins. ProA-tagged Pre1 and Rpn11 proteasome subunits were detected by the direct interaction of ProA with IgG. Sem1 antibody specificity was confirmed using extracts from asem1Δ strain as negative control; the additional Sem1-specific bands of lower apparent molecular weight cofractionating with the proteasome were not further characterized.
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