Assembly of the Drosophila 26 S proteasome is accompanied by extensive subunit rearrangements.
The subunit contacts in the regulatory complex of the Drosophila 26 S proteasome were studied through the cross-linking of closely spaced subunits of the complex, and analysis of the cross-linking pattern in an immunoblot assay with the use of subunit-specific monoclonal antibodies. The cross-linking pattern of the purified 26 S proteasome exhibits significant differences as compared with that of the purified free regulatory complex. It is shown that the observed differences are due to extensive rearrangement of the subunit contacts accompanying the assembly of the 26 S proteasome from the regulatory complex and the 20 S proteasome. Cross-linking studies and electron microscopic examinations revealed that these changes are reversible and follow the assembly or the disassembly of the 26 S proteasome. Although the majority of the changes observed in the subunit contacts affected the hexameric ring of the ATPase subunits, the alterations extended over the whole of the regulatory complex, affecting subunit contacts even in the lid subcomplex. Changes in the subunit contacts, similar to those in the regulatory complex, were detected in the 20 S proteasome. These observations indicate that the assembly of the 26 S proteasome is not simply a passive docking of two rigid subcomplexes. In the course of the assembly, the interacting subcomplexes mutually rearrange their structures so as to create the optimal conformation required for the assembly and the proper functioning of the 26 S proteasome.
Kurucz
Eva
E
Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary.
Andó
István
I
Sümegi
Máté
M
Hölzl
Harald
H
Kapelari
Barbara
B
Baumeister
Wolfgang
W
Udvardy
Andor
A
D000818:Animals; D015153:Blotting, Western; D004330:Drosophila / Q000196:embryology; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000201:enzymology; D008854:Microscopy, Electron; D010447:Peptide Hydrolases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease