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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.


CP undergoes affinity switch upon maturation(a) Affinity-resin bound purified CP was reconstituted with heterologously expressed His-tagged Pba1-Pba2 dimer. Samples were washed with buffer containing the indicated NaCl concentrations. Next, samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for the indicated proteins. (b) YFP-tagged CP was first reconstituted with 5 fold molar excess of Pba1-Pba2 and challenged with different amounts of purified RP (molar excess over CP indicated). Samples were resolved on a native gel to determine complex composition in samples: Top panel, proteolytic active complexes were visualized using in gel suc-LLVY-AMC hydrolytic activity assay. Middle panel, native gel was immunoblotted for the RP subunit Rpn1. Lower panel, native gel was analyzed for YFP signal. (c-f) Immature CP or 26S proteasome from wildtype or indicated strains were purified, split into different samples and washed with buffer containing the indicated NaCl concentrations. Samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for indicated proteins. The asterisk indicates an IgG resin-derived band.
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Figure 3: CP undergoes affinity switch upon maturation(a) Affinity-resin bound purified CP was reconstituted with heterologously expressed His-tagged Pba1-Pba2 dimer. Samples were washed with buffer containing the indicated NaCl concentrations. Next, samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for the indicated proteins. (b) YFP-tagged CP was first reconstituted with 5 fold molar excess of Pba1-Pba2 and challenged with different amounts of purified RP (molar excess over CP indicated). Samples were resolved on a native gel to determine complex composition in samples: Top panel, proteolytic active complexes were visualized using in gel suc-LLVY-AMC hydrolytic activity assay. Middle panel, native gel was immunoblotted for the RP subunit Rpn1. Lower panel, native gel was analyzed for YFP signal. (c-f) Immature CP or 26S proteasome from wildtype or indicated strains were purified, split into different samples and washed with buffer containing the indicated NaCl concentrations. Samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for indicated proteins. The asterisk indicates an IgG resin-derived band.

Mentions: Since CP does not degrade Pba1-Pba2, RP must be able to displace it directly or with the help of other factors. Previous work using surface plasmon resonance had shown that the interaction between mature CP and Pba1-Pba2 is salt-labile8. We also observe that this interaction can be disrupted with low salt concentrations when we reconstituted E. coli purified Pba1-Pba2 onto resin-bound CP purified from yeast (Fig. 3a). Using bio-layer interferometry, we measured a KD of ~ 1 μM for Pba1-Pba2 with mature CP in 50 mM NaCl (Supplementary Figure 2a), which is similar to the 3 μM reported by Stadtmueller et al. under physiological salt concentrations8. Considering the reported nanomolar range affinities between the RP subunit containing complexes and CP21,30, these findings suggest that the RP will likely displace Pba1-Pba2 from the mature CP.


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)

CP undergoes affinity switch upon maturation(a) Affinity-resin bound purified CP was reconstituted with heterologously expressed His-tagged Pba1-Pba2 dimer. Samples were washed with buffer containing the indicated NaCl concentrations. Next, samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for the indicated proteins. (b) YFP-tagged CP was first reconstituted with 5 fold molar excess of Pba1-Pba2 and challenged with different amounts of purified RP (molar excess over CP indicated). Samples were resolved on a native gel to determine complex composition in samples: Top panel, proteolytic active complexes were visualized using in gel suc-LLVY-AMC hydrolytic activity assay. Middle panel, native gel was immunoblotted for the RP subunit Rpn1. Lower panel, native gel was analyzed for YFP signal. (c-f) Immature CP or 26S proteasome from wildtype or indicated strains were purified, split into different samples and washed with buffer containing the indicated NaCl concentrations. Samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for indicated proteins. The asterisk indicates an IgG resin-derived band.
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Related In: Results  -  Collection

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Figure 3: CP undergoes affinity switch upon maturation(a) Affinity-resin bound purified CP was reconstituted with heterologously expressed His-tagged Pba1-Pba2 dimer. Samples were washed with buffer containing the indicated NaCl concentrations. Next, samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for the indicated proteins. (b) YFP-tagged CP was first reconstituted with 5 fold molar excess of Pba1-Pba2 and challenged with different amounts of purified RP (molar excess over CP indicated). Samples were resolved on a native gel to determine complex composition in samples: Top panel, proteolytic active complexes were visualized using in gel suc-LLVY-AMC hydrolytic activity assay. Middle panel, native gel was immunoblotted for the RP subunit Rpn1. Lower panel, native gel was analyzed for YFP signal. (c-f) Immature CP or 26S proteasome from wildtype or indicated strains were purified, split into different samples and washed with buffer containing the indicated NaCl concentrations. Samples were resolved on SDS-PAGE and stained with CBB or immunoblotted for indicated proteins. The asterisk indicates an IgG resin-derived band.
Mentions: Since CP does not degrade Pba1-Pba2, RP must be able to displace it directly or with the help of other factors. Previous work using surface plasmon resonance had shown that the interaction between mature CP and Pba1-Pba2 is salt-labile8. We also observe that this interaction can be disrupted with low salt concentrations when we reconstituted E. coli purified Pba1-Pba2 onto resin-bound CP purified from yeast (Fig. 3a). Using bio-layer interferometry, we measured a KD of ~ 1 μM for Pba1-Pba2 with mature CP in 50 mM NaCl (Supplementary Figure 2a), which is similar to the 3 μM reported by Stadtmueller et al. under physiological salt concentrations8. Considering the reported nanomolar range affinities between the RP subunit containing complexes and CP21,30, these findings suggest that the RP will likely displace Pba1-Pba2 from the mature CP.

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.