PA28 activator protein forms regulatory caps on proteasome stacked rings.
The proteasome is a 700,000 dalton proteolytic complex found in eukaryotes and, in a simpler form, in archaebacteria. Its distinctive architecture consists of a stack of four rings, each containing approximately six to eight 21,000 to 35,000 dalton subunits. In this report, we describe the use of electron microscopy of negatively stained specimens, including alignment and averaging of multiple digitized images, to investigate the complexes formed between eukaryotic proteasomes and a recently-isolated 28,000 dalton eukaryotic proteasome activator, PA28. We find that purified PA28, which was previously shown to have a native molecular mass consistent with oligomerization of the PA28 polypeptide, occurs as a ring of variably-positioned protein subunits. In one set of images, these subunits appear to be tethered to a central hub. When incubated with proteasomes, PA28 forms oligomeric regulatory caps on both ends of eukaryotic proteasomes, with the proteasome outer rings serving as scaffolds to which the bases of the regulatory caps are attached. The base of each cap consists of a thickened protein mass, so that the base is wider than the tip of the cap. A stain-filled channel penetrates into each cap from its tip, indicating that the protein subunits tend to separate somewhat at the tip. The caps are about 10 to 11 nm wide at the base and 7 to 8 nm long from base to tip. This is the first direct visualization of the interaction between proteasomes and a purified, functionally characterized protein modulator of proteasome activity, and it gives the first insight as to the particle geometry through which activation by PA28 must occur. The capping at both ends implies the existence of an underlying 2-fold symmetry (or pseudosymmetry) in the eukaryotic proteasome, suggesting that proteasomes may consist of two identical or quasi-identical structural units which interact at a central interface.
Gray
C W
CW
Program in Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688.
Slaughter
C A
CA
DeMartino
G N
GN
D000818:Animals; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000648:ultrastructure*; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism; D046911:Macromolecular Substances; D008854:Microscopy, Electron / Q000379:methods; D008961:Models, Structural; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000648:ultrastructure*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000302:isolation & purification / Q000648:ultrastructure*
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011506:Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex