Biochemical purification of distinct proteasome subsets.
Proteasomes are intricate cellular proteases that are able to degrade many protein and peptide substrates in vitro. These particles are structurally complex; they are assembled from at least 14 small molecular mass polypeptide subunits to form mature 20S proteasomes. Recently, we demonstrated that proteasome subsets may be discriminated by serological criteria, and have found that subtle differences in the subunit composition of proteasomes can alter their cleavage specificity. Proteasome structural complexity is further enhanced when some proteasomes associate with additional proteins to form a 26S ATP- and ubiquitin-dependent protease. Herein we confirm the existence of distinct cellular forms of proteasomes, and show that they differ in their hydrophobic characteristics. We have reproducibly purified, using solely biochemical techniques, distinct proteasome subsets similar to the serologically defined LMP2+ and LMP2- proteasomes. These proteasome subsets differ in their expression of at least three polypeptides, and both lack several additional polypeptides as compared to the serologically defined LMP2+ and LMP2- proteasomes. Finally, we demonstrate that these structurally unique proteasomes differ in their capacity to cleave a defined panel of fluorogenic peptide substrates. It appears that mammalian cells might recruit and modify proteasomes to perform distinct cellular tasks.
Brown
M G
MG
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond.
Monaco
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003374:Coumarins / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D013379:Substrate Specificity
D003374:Coumarins; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009842:Oligopeptides; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex