Structure of the Blm10-20 S proteasome complex by cryo-electron microscopy. Insights into the mechanism of activation of mature yeast proteasomes.
The 20 S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20 S proteasome: the 19 S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10-mature yeast 20 S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10-20 S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20 S particle and contacts all the proteasome alpha subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the alpha rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.
Iwanczyk
Jack
J
Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada.
Sadre-Bazzaz
Kianoush
K
Ferrell
Katherine
K
Kondrashkina
Elena
E
Formosa
Timothy
T
Hill
Christopher P
CP
Ortega
Joaquin
J
D000818:Animals; D002417:Cattle; D020285:Cryoelectron Microscopy; D004249:DNA Damage; D004789:Enzyme Activation; D008958:Models, Molecular; D009097:Multienzyme Complexes; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C500597:Blm10 protein, S cerevisiae; D009097:Multienzyme Complexes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex