Electron microscopic evidence in support of alpha-solenoid models of proteasomal subunits Rpn1 and Rpn2.
Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal alpha-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each approximately 40 residues long and containing two alpha-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly alpha-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the alpha-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures.
Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Grégory
G
Effantin
Rina
R
Rosenzweig
Michael H
MH
Glickman
Alasdair C
AC
Steven
D002850:Chromatography, Gel; D002942:Circular Dichroism; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics
C499663:RPN1 protein, S cerevisiae; C096166:RPN2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex