Characterization of membrane-associated proteasomes in WB rat liver epithelial cells.
Although proteasomes are mainly located in the cytosol, it is known that significant amounts are also associated with endoplasmic reticulum (ER) membranes where they may play a role in the degradation of specific ER membrane proteins. The present studies were undertaken to compare ER and cytosolic proteasomal activities in WB rat liver cells. N-Heptyl-beta-thioglucopyranoside (HTG) extracts of membrane or cytosol fractions were chromatographed in glycerol/ATP buffers on size-exclusion and ion-exchange columns and the elution profiles of proteasomal peptidase activity and immunoreactive components of the 20S complex, 19S complex, and PA28 were compared. Cytosol fractions showed a single peak of chymotrypsin-like peptidase activity (Cht-L), which was inhibited completely by 5 microM 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 activity observed in cytosol fractions. However, the second peak was stimulated by SDS and was LC-insensitive (5 microM) and contained trypsin-like (T-L) and peptide-glutamyl peptidase (PGPH) but no cathepsin or calcium-activated protease activities. PA28 activator protein was present in both membrane and cytosol fractions. Thus, the principal difference between cytosolic and membrane activity was that the latter fractions contained a novel membrane-associated LC-insensitive protease(s) catalyzing three of the major peptidase activities of the proteasome.
Khan
M T
MT
Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107, USA.
Joseph
S K
SK
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D000837:Anion Exchange Resins / Q000737:chemistry; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002403:Cathepsins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry; D051381:Rats; D012117:Resins, Synthetic; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000837:Anion Exchange Resins; D018797:Cell Cycle Proteins; D003902:Detergents; D009097:Multienzyme Complexes; D011506:Proteins; C490050:Psme1 protein, rat; C490052:Psme2 protein, rat; D012117:Resins, Synthetic; C067713:lactacystin; D012967:Sodium Dodecyl Sulfate; C042855:Mono Q; D002403:Cathepsins; D010450:Endopeptidases; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium; D000111:Acetylcysteine