Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi.
Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpnl (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpnl are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpnl in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.
Zou
C B
CB
Department of Parasitology, Juntendo University School of Medicine, Tokyo, Japan.
Nakajima-Shimada
J
J
Nara
T
T
Aoki
T
T
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014158:Transcription, Genetic; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D005656:Fungal Proteins; D009097:Multienzyme Complexes; D011506:Proteins; D015800:Protozoan Proteins; C425294:Rpn1 protein, Trypanosoma; D029701:Saccharomyces cerevisiae Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex