Molecular cloning of two types of cDNA encoding subunit RC6-I of rat proteasomes.
A new subunit, named RC6-I, of the rat 20 S proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by Edman degradation. Amplification of cDNAs encoding RC6-I by the polymerase chain reaction (PCR) technique revealed two types of cDNA, tentatively designated as RC6-IL and RC6-IS in order of size. The nucleotide sequences of the two cDNAs are identical except that RC6-IL contains an insertion of 18 nucleotides in the coding region compared with RC6-IS. The polypeptide predicted from the open reading frame of RC6-IS cDNA consists of 248 amino acid residues with a calculated molecular weight of 27,783. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology analysis showed that RC6-I belongs to the alpha-type subfamily of the proteasome gene family, which shows similarity to the alpha-subunit of the archaebacterium Thermoplasma acidophilum proteasome, and that the 18 nucleotide insert, encoding six amino acid residues, VVASVS, appears to be unique to RC6-IL, because this motif has not been conserved in any other alpha-type subunit. By reverse transcription (RT)-PCR analysis, the mRNAs for both RC6-IL and RC6-IS were found in all the rat tissues examined. These results suggest that proteasomes are present as a heterogeneous population, possibly for acquisition of diversity of functions.
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Institute for Enzyme Research, University of Tokushima, Japan.
Tomita
Y
Y
Tokunaga
F
F
Liang
T J
TJ
Noda
C
C
Ichihara
A
A
Tanaka
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D010450:Endopeptidases; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D012333:RNA, Messenger; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex