Characterization of membrane-associated proteasomes in WB rat liver epithelial cells

Although proteasomes are mainly located in the cytosol, it is known that si gnificant amounts are also associated with endoplasmic reticulum (ER) membr anes where they may play a role in the degradation of specific ER membrane proteins. The present studies were undertaken to compare ER and cytosolic p roteasomal activities in WE rat liver cells, N-Heptyl-beta -thioglucopyrano side (HTG) extracts of membrane or cytosol fractions were chromatographed i n glycerol/ATP buffers on size-exclusion and ion-exchange columns and the e lution profiles of proteasomal peptidase activity and immunoreactive compon ents of the 20S complex, 19S complex, and PA28 were compared. Cytosol fract ions showed a single peak of chymotrypsinlike peptidase activity (Cht-L), w hich was inhibited completely by 5 muM lactacystin (LC) or SDS (0.03%) and corresponded to 26S proteasomes based upon the presence of both 20S and 19S components. By comparison, membrane fractions contained two major peaks of Cht-L activity. The first peak shared the same properties as the peak acti vity observed in cytosol fractions, However, the second peak was stimulated by SDS and was LC-insensitive (5 muM) and contained trypsin-like (T-L) and peptide-glutamyl peptidase (PGPH) but no cathepsin or calcium-activated pr otease activities. PA28 activator protein was present in both membrane and cytosol fractions, Thus, the principal difference between cytosolic and mem brane activity was that the latter fractions contained a novel membrane-ass ociated LC-insensitive protease(s) catalyzing three of the major peptidase activities of the proteasome, (C) 2001 Academic Press.