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Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association.

Wani PS, Rowland MA, Ondracek A, Deeds EJ, Roelofs J - Nat Commun (2015)

Bottom Line: Changes in the CP that occur on maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone.Mathematical modelling indicates that this 'affinity switch' mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates.Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Biochemistry Group, Department of Biochemistry and Molecular Biophysics, Kansas State University, 336 Ackert Hall, Manhattan, Kansas 66506, USA.

ABSTRACT
Proteasome assembly is a complex process, requiring 66 subunits distributed over several subcomplexes to associate in a coordinated fashion. Ten proteasome-specific chaperones have been identified that assist in this process. For two of these, the Pba1-Pba2 dimer, it is well established that they only bind immature core particles (CPs) in vivo. In contrast, the regulatory particle (RP) utilizes the same binding surface but only interacts with the mature CP in vivo. It is unclear how these binding events are regulated. Here, we show that Pba1-Pba2 binds tightly to the immature CP, preventing RP binding. Changes in the CP that occur on maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone. Mathematical modelling indicates that this 'affinity switch' mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates. Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis.

No MeSH data available.


Related in: MedlinePlus

Pba1-Pba2 are required for efficient incorporation of α5 and α6(a) Immature CP from wildtype strain was purified and resolved on SDS-PAGE. Two bands that were consistently absent or reduced in a pba1Δ background (See Fig. 1c, 3c, and 3d) were excised and submitted to mass spectrometry analysis for identification (see Table 1). Band 1 was identified as Pba1 and Pba2, while α6 was the major component of Band 2, consistent with previous assignments15,26,43. (b) Unique peptides from the mass spectrometry analyses in (a) that cover regions of the pro-peptides of β1, β2, and β5 are underlined with the pro-peptide sequence in bold. (c) Purified mature or immature CP from indicated strains were resolved by two-dimensional gel electrophoresis using a non-linear ZOOM® IPG strip (pH 3–10) followed by SDS-PAGE. Gels were stained with CBB. Proteins spots were assigned based on immunoblotting (Supplementary Figure 3a), mass spectrometry analyses (underlined and Supplementary Figure 3b), and previous assignments26,43.
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Figure 4: Pba1-Pba2 are required for efficient incorporation of α5 and α6(a) Immature CP from wildtype strain was purified and resolved on SDS-PAGE. Two bands that were consistently absent or reduced in a pba1Δ background (See Fig. 1c, 3c, and 3d) were excised and submitted to mass spectrometry analysis for identification (see Table 1). Band 1 was identified as Pba1 and Pba2, while α6 was the major component of Band 2, consistent with previous assignments15,26,43. (b) Unique peptides from the mass spectrometry analyses in (a) that cover regions of the pro-peptides of β1, β2, and β5 are underlined with the pro-peptide sequence in bold. (c) Purified mature or immature CP from indicated strains were resolved by two-dimensional gel electrophoresis using a non-linear ZOOM® IPG strip (pH 3–10) followed by SDS-PAGE. Gels were stained with CBB. Proteins spots were assigned based on immunoblotting (Supplementary Figure 3a), mass spectrometry analyses (underlined and Supplementary Figure 3b), and previous assignments26,43.

Mentions: Upon purification of immature CP from a pba1Δ strain we noticed a reduced level of a specific proteasome core particle subunit after SDS-PAGE analysis (compare Fig. 3c and 3d). Based on the running behavior, previous assignments by other labs26,43 and our own mass spectrometry analysis (Fig. 4a, 4b, and Table 1) we determined this was the subunit α6. This suggests that there is a reduced level of α6 present in immature proteasomes in the absence of Pba1. Additional mass spectrometry analysis suggested that besides α6 there might also be reduced levels of α5. To test this, we ran 2D gels of mature CP from wildtype and immature CP from wildtype and pba1Δ strains (Fig. 4c and Supplementary Figure 3). These gels confirmed the substantial reduction in levels of α5 and α6 from immature CP purified from pba1Δ strains. Pba1 thus likely has a role in efficient incorporation of α5 and α6, similar to Pba3-Pba4, which has a role in controlling α3 (Pre9) and α4 (Pre6) incorporation. However, α5 and α6 are essential subunits of the proteasome, making a potential role for Pba1-Pba2 in formation of alternative proteasomes, as was proposed for Pba3-Pba43, unlikely. Considering the thermodynamic stability of rings once they have formed44-46, the reduced level of these two subunits most likely indicates a slow incorporation of these subunits into the α ring in a pba1Δ strain. However, an alternative or additional role of Pba1-Pba2 might be the stabilization of α5 and α6 once they have been incorporated into the α ring, in particular since α5 and α6 appear to be absent in the high salt wash of the pba1-ΔCT3 pba2-ΔCT3 strain (Fig. 3F).


Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association.

Wani PS, Rowland MA, Ondracek A, Deeds EJ, Roelofs J - Nat Commun (2015)

Pba1-Pba2 are required for efficient incorporation of α5 and α6(a) Immature CP from wildtype strain was purified and resolved on SDS-PAGE. Two bands that were consistently absent or reduced in a pba1Δ background (See Fig. 1c, 3c, and 3d) were excised and submitted to mass spectrometry analysis for identification (see Table 1). Band 1 was identified as Pba1 and Pba2, while α6 was the major component of Band 2, consistent with previous assignments15,26,43. (b) Unique peptides from the mass spectrometry analyses in (a) that cover regions of the pro-peptides of β1, β2, and β5 are underlined with the pro-peptide sequence in bold. (c) Purified mature or immature CP from indicated strains were resolved by two-dimensional gel electrophoresis using a non-linear ZOOM® IPG strip (pH 3–10) followed by SDS-PAGE. Gels were stained with CBB. Proteins spots were assigned based on immunoblotting (Supplementary Figure 3a), mass spectrometry analyses (underlined and Supplementary Figure 3b), and previous assignments26,43.
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Figure 4: Pba1-Pba2 are required for efficient incorporation of α5 and α6(a) Immature CP from wildtype strain was purified and resolved on SDS-PAGE. Two bands that were consistently absent or reduced in a pba1Δ background (See Fig. 1c, 3c, and 3d) were excised and submitted to mass spectrometry analysis for identification (see Table 1). Band 1 was identified as Pba1 and Pba2, while α6 was the major component of Band 2, consistent with previous assignments15,26,43. (b) Unique peptides from the mass spectrometry analyses in (a) that cover regions of the pro-peptides of β1, β2, and β5 are underlined with the pro-peptide sequence in bold. (c) Purified mature or immature CP from indicated strains were resolved by two-dimensional gel electrophoresis using a non-linear ZOOM® IPG strip (pH 3–10) followed by SDS-PAGE. Gels were stained with CBB. Proteins spots were assigned based on immunoblotting (Supplementary Figure 3a), mass spectrometry analyses (underlined and Supplementary Figure 3b), and previous assignments26,43.
Mentions: Upon purification of immature CP from a pba1Δ strain we noticed a reduced level of a specific proteasome core particle subunit after SDS-PAGE analysis (compare Fig. 3c and 3d). Based on the running behavior, previous assignments by other labs26,43 and our own mass spectrometry analysis (Fig. 4a, 4b, and Table 1) we determined this was the subunit α6. This suggests that there is a reduced level of α6 present in immature proteasomes in the absence of Pba1. Additional mass spectrometry analysis suggested that besides α6 there might also be reduced levels of α5. To test this, we ran 2D gels of mature CP from wildtype and immature CP from wildtype and pba1Δ strains (Fig. 4c and Supplementary Figure 3). These gels confirmed the substantial reduction in levels of α5 and α6 from immature CP purified from pba1Δ strains. Pba1 thus likely has a role in efficient incorporation of α5 and α6, similar to Pba3-Pba4, which has a role in controlling α3 (Pre9) and α4 (Pre6) incorporation. However, α5 and α6 are essential subunits of the proteasome, making a potential role for Pba1-Pba2 in formation of alternative proteasomes, as was proposed for Pba3-Pba43, unlikely. Considering the thermodynamic stability of rings once they have formed44-46, the reduced level of these two subunits most likely indicates a slow incorporation of these subunits into the α ring in a pba1Δ strain. However, an alternative or additional role of Pba1-Pba2 might be the stabilization of α5 and α6 once they have been incorporated into the α ring, in particular since α5 and α6 appear to be absent in the high salt wash of the pba1-ΔCT3 pba2-ΔCT3 strain (Fig. 3F).

Bottom Line: Changes in the CP that occur on maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone.Mathematical modelling indicates that this 'affinity switch' mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates.Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Biochemistry Group, Department of Biochemistry and Molecular Biophysics, Kansas State University, 336 Ackert Hall, Manhattan, Kansas 66506, USA.

ABSTRACT
Proteasome assembly is a complex process, requiring 66 subunits distributed over several subcomplexes to associate in a coordinated fashion. Ten proteasome-specific chaperones have been identified that assist in this process. For two of these, the Pba1-Pba2 dimer, it is well established that they only bind immature core particles (CPs) in vivo. In contrast, the regulatory particle (RP) utilizes the same binding surface but only interacts with the mature CP in vivo. It is unclear how these binding events are regulated. Here, we show that Pba1-Pba2 binds tightly to the immature CP, preventing RP binding. Changes in the CP that occur on maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone. Mathematical modelling indicates that this 'affinity switch' mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates. Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis.

No MeSH data available.


Related in: MedlinePlus