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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 prevents RP association with immature CP(a) Proteasomes were purified from indicated strains, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB). (b) Native gel analysis of samples from (A) stained in gel with the proteasome substrate suc-LLVY-AMC (left) or stained with CBB. (c) 26S proteasome or immature CP were affinity-purified using ProA-tagged β4 or TAP-tagged Ump1 respectively. Purified complexes were resolved on SDS-PAGE and stained with CBB (top) or immunoblotted for Pba1-Pba2, indicated proteasome subunits (CP indicates core and RP indicates regulatory particle subunits), and Ecm29. The asterisk marks a non-specific band. (d) 26S proteasome and CP were purified using standard protocols. Immature CP was purified and washed with buffers containing the indicated NaCl concentrations. Samples were resolved on a native gel and stained with either suc-LLVY-AMC (top) or with CBB (bottom).
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Figure 1: Pba1-Pba2 prevents RP association with immature CP(a) Proteasomes were purified from indicated strains, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB). (b) Native gel analysis of samples from (A) stained in gel with the proteasome substrate suc-LLVY-AMC (left) or stained with CBB. (c) 26S proteasome or immature CP were affinity-purified using ProA-tagged β4 or TAP-tagged Ump1 respectively. Purified complexes were resolved on SDS-PAGE and stained with CBB (top) or immunoblotted for Pba1-Pba2, indicated proteasome subunits (CP indicates core and RP indicates regulatory particle subunits), and Ecm29. The asterisk marks a non-specific band. (d) 26S proteasome and CP were purified using standard protocols. Immature CP was purified and washed with buffers containing the indicated NaCl concentrations. Samples were resolved on a native gel and stained with either suc-LLVY-AMC (top) or with CBB (bottom).

Mentions: As mentioned above, Pba1-Pba2 and Blm10 likely compete with each other for binding to the CP α ring. Consistent with this idea, upon purification of proteasomes from pba1Δ, pba2Δ or pba1Δpba2Δ yeast strains we observed increased levels of Blm10 (Fig. 1a, 1b, and Supplementary Figure 1a). However, in vivo Pba1-Pba2 is normally only found on the immature CP, and it has been postulated that one function of Pba1-Pba2 could be to prevent RP from binding to immature CP8,9,35,36. To test this hypothesis, we first affinity-purified immature CP using TAP-tagged Ump1 (Fig. 1c and 1d). Mass spectrometry analyses confirmed that these purifications contained all seven α subunits in addition to β1 to β5, Pba1, Pba2 and Ump1 (Supplementary Table 1). β6 (Pre7) and β7 (Pre4) were undetectable, as has been observed before37. The presence of Pba1-Pba2 was also confirmed by immunoblotting (Fig. 1c and Supplementary Figure 1b). Propeptides for β1, β2, and β5 were detected by immunoblotting or mass spectrometry (Fig. 1c and Supplementary Table 1). Taken together, these data indicate we purified a form of CP that has not matured yet.


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 prevents RP association with immature CP(a) Proteasomes were purified from indicated strains, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB). (b) Native gel analysis of samples from (A) stained in gel with the proteasome substrate suc-LLVY-AMC (left) or stained with CBB. (c) 26S proteasome or immature CP were affinity-purified using ProA-tagged β4 or TAP-tagged Ump1 respectively. Purified complexes were resolved on SDS-PAGE and stained with CBB (top) or immunoblotted for Pba1-Pba2, indicated proteasome subunits (CP indicates core and RP indicates regulatory particle subunits), and Ecm29. The asterisk marks a non-specific band. (d) 26S proteasome and CP were purified using standard protocols. Immature CP was purified and washed with buffers containing the indicated NaCl concentrations. Samples were resolved on a native gel and stained with either suc-LLVY-AMC (top) or with CBB (bottom).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4380239&req=5

Figure 1: Pba1-Pba2 prevents RP association with immature CP(a) Proteasomes were purified from indicated strains, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB). (b) Native gel analysis of samples from (A) stained in gel with the proteasome substrate suc-LLVY-AMC (left) or stained with CBB. (c) 26S proteasome or immature CP were affinity-purified using ProA-tagged β4 or TAP-tagged Ump1 respectively. Purified complexes were resolved on SDS-PAGE and stained with CBB (top) or immunoblotted for Pba1-Pba2, indicated proteasome subunits (CP indicates core and RP indicates regulatory particle subunits), and Ecm29. The asterisk marks a non-specific band. (d) 26S proteasome and CP were purified using standard protocols. Immature CP was purified and washed with buffers containing the indicated NaCl concentrations. Samples were resolved on a native gel and stained with either suc-LLVY-AMC (top) or with CBB (bottom).
Mentions: As mentioned above, Pba1-Pba2 and Blm10 likely compete with each other for binding to the CP α ring. Consistent with this idea, upon purification of proteasomes from pba1Δ, pba2Δ or pba1Δpba2Δ yeast strains we observed increased levels of Blm10 (Fig. 1a, 1b, and Supplementary Figure 1a). However, in vivo Pba1-Pba2 is normally only found on the immature CP, and it has been postulated that one function of Pba1-Pba2 could be to prevent RP from binding to immature CP8,9,35,36. To test this hypothesis, we first affinity-purified immature CP using TAP-tagged Ump1 (Fig. 1c and 1d). Mass spectrometry analyses confirmed that these purifications contained all seven α subunits in addition to β1 to β5, Pba1, Pba2 and Ump1 (Supplementary Table 1). β6 (Pre7) and β7 (Pre4) were undetectable, as has been observed before37. The presence of Pba1-Pba2 was also confirmed by immunoblotting (Fig. 1c and Supplementary Figure 1b). Propeptides for β1, β2, and β5 were detected by immunoblotting or mass spectrometry (Fig. 1c and Supplementary Table 1). Taken together, these data indicate we purified a form of CP that has not matured yet.

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