title: Conformational dynamics of the Rpt6 ATPase in proteasome assembly and Rpn14 binding.
abstract: Juxtaposed to either or both ends of the proteasome core particle (CP) can exist a 19S regulatory particle (RP) that recognizes and prepares ubiquitinated proteins for proteolysis. RP triphosphatase proteins (Rpt1-Rpt6), which are critical for substrate translocation into the CP, bind chaperone-like proteins (Hsm3, Nas2, Nas6, and Rpn14) implicated in RP assembly. We used NMR and other biophysical methods to reveal that S. cerevisiae Rpt6's C-terminal domain undergoes dynamic helix-coil transitions enabled by helix-destabilizing glycines within its two most C-terminal α helices. Rpn14 binds selectively to Rpt6's four-helix bundle, with surprisingly high affinity. Loss of Rpt6's partially unfolded state by glycine substitution (Rpt6 G³⁶⁰,³⁸⁷A) disrupts holoenzyme formation in vitro, an effect enhanced by Rpn14. S. cerevisiae lacking Rpn14 and incorporating Rpt6 G³⁶⁰,³⁸⁷A demonstrate hallmarks of defective proteasome assembly and synthetic growth defects. Rpt4 and Rpt5 exhibit similar exchange, suggesting that conserved structural heterogeneity among Rpt proteins may facilitate RP-CP assembly.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Ehlinger
authors.forename: Soyeon
authors.initials: S
authors.lastname: Park
authors.forename: Amr
authors.initials: A
authors.lastname: Fahmy
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Lary
authors.forename: James L
authors.initials: JL
authors.lastname: Cole
authors.forename: Daniel
authors.initials: D
authors.lastname: Finley
authors.forename: Kylie J
authors.initials: KJ
authors.lastname: Walters
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex; C542569:Rpn14 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C069670:RPT6 protein, S cerevisiae; D005998:Glycine
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