Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease.
The multicatalytic protease (MCP) can be activated by two distinct multisubunit complexes. One is the regulatory component of the 26 S protease, which contains at least 15 distinct subunits. The other is a hexameric activator composed of 31- and 29-kDa subunits. A cDNA for the smaller subunit has been cloned and sequenced. The cDNA encodes a protein of 249 amino acids. Embedded between sequences typical of globular protein domains is a stretch of 28 "alternating" lysine and glutamic acid residues. Similar regions, which we call KEKE motifs, are also found in two MCP subunits, in subunit 12 of the 26 S protease and in a variety of chaperonins including hsp90, hsp70, and calnexin. Expression of the activator cDNA in Escherichia coli produced a functional protein virtually indistinguishable from MCP activator purified directly from red blood cells. The recombinant protein formed three isoelectric species on two-dimensional polyacrylamide gel electrophoresis, and it reacted with antibodies to red blood cell activator. Recombinant activator also bound the multicatalytic protease and stimulated cleavage at the carboxyl terminus of hydrophobic or charged residues. Synthesis of the activator subunit was induced by gamma interferon treatment of HeLa cells. These last two findings have implications for antigen presentation by class I major histocompatibility receptors.
Realini
C
C
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Dubiel
W
W
Pratt
G
G
Ferrell
K
K
Rechsteiner
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D018076:DNA, Complementary; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011506:Proteins; D011994:Recombinant Proteins; C088683:multicatalytic protease activator; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex