title: Characterization of membrane-associated proteasomes in WB rat liver epithelial cells.
abstract: 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.
authors.affiliation: Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Khan
authors.forename: S K
authors.initials: SK
authors.lastname: Joseph
mesh_terms: 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
chemical_list: 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