Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei.
We have determined peptide sequences of three Trypanosoma brucei proteasome subunit proteins by mass spectrometry of tryptic digests of the proteins purified by two-dimensional (2-D) polyacrylamide gel electrophoresis. Three genes identified by the sequence of their cDNA encode the peptides identified in these three proteins. The three proteins predicted from the gene sequences have significant similarity to other known proteasome subunits and represent an alpha6 type subunit (TbPSA6), and two beta-type subunits belonging to the beta1-type (TbPSB1) and beta2 type (TbPSB2). The sequences of both beta-subunits predict formation of catalytically active subunits through proteolytic processing. The prediction is supported by the presence in each of the two beta-subunits of a tryptic peptide that has the correctly processed N-terminus that creates the threonine nucleophile of the mature protein. This peptide cannot be generated by trypsin because of the required cleavage of a glycine-threonine bond. It is thus likely that there are at least two catalytically active beta-subunits, TbPSB1 and TbPSB2, present in the mature 20S proteasome from T. brucei.
Huang
L
L
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Shen
M
M
Chernushevich
I
I
Burlingame
A L
AL
Wang
C C
CC
Robertson
C D
CD
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D017125:Genes, Protozoan; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017422:Sequence Analysis, DNA; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex