Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) 
membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome 
(ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium 
cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE 
analysis revealed that the purified ERa and ERb proteasomes were composed of 
multiple subunits similar to the cytosolic 20S proteasome. However, 
electrophoretic, structural and immunochemical differences between the ERa, ERb 
and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) 
PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate 
extract of the trypsin-treated microsomal fraction yielded a trypsin-modified 
form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no 
ERa proteasome was obtained from the 0.0125% sodium cholate extract of the 
trypsin-treated microsomes, suggesting that ERa and ERb are ER 
membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and 
cytosolic 20S proteasomes exhibited similar specificities as to peptide 
hydrolyzing activity, although differences in their activities were noted in the 
presence of SDS and phospholipid. With respect to the proteolysis of protein 
substrates, only the ERb proteasome cleaved beta-casein, and it also degraded 
reduced and carboxymethylated lysozyme considerably faster than the cytosolic 
20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb 
proteasomes may play roles in intracellular proteolysis distinct from that of 
the cytosolic 20S proteasome.