cDNA cloning and functional analysis of the p97 subunit of the 26S proteasome, a polypeptide identical to the type-1 tumor-necrosis-factor-receptor-associated protein-2/55.11.
Molecular cloning of cDNA for a new regulatory subunit, designated p97, of the human 26S proteasome showed that the polypeptide consists of 908 amino acid residues with a calculated molecular mass of 100184 Da and an isoelectric point of 4.94. Computer analysis showed that p97 is very similar to type-1 tumor-necrosis-factor (TNF)-receptor-associated protein (TRAP)-2 and 55.11, both of which were identified recently as binding proteins of the cytoplasmic domain of type-1 TNF receptor by yeast two-hybrid screening. This finding suggests that the 26S proteasome might serve as a mediator molecule in the TNF signaling pathway in cells. Computer-assisted similarity analysis also revealed the high sequence similarity of p97 with a yeast protein whose function is yet unknown, the gene for which is here termed NAS1 (non-ATPase subunit 1). Disruption of NAS1 resulted in several phenotypes, including lethality and temperature-sensitive growth, depending on the genetic background of the cells used. The human p97 cDNA suppressed the growth defect of nas1 disruptant cells, when expressed from single-copy or multi-copy vectors, indicating that p97 is functionally equivalent to yeast Nas1p. Culturing of the temperature-sensitive nas1 cells at the restrictive temperature promoted the accumulation polyubiquitinated cellular proteins, implying that the 26S proteasome requires a functional Nas1p subunit for ubiquitin-dependent proteolysis. These results indicate that p97/Nas1p plays an important regulatory role in the function of the 26S proteasome.
Tsurumi
C
C
Institute for Enzyme Research, University of Tokushima, Japan.
Shimizu
Y
Y
Saeki
M
M
Kato
S
S
Demartino
G N
GN
Slaughter
C A
CA
Fujimuro
M
M
Yokosawa
H
H
Yamasaki
M
M
Hendil
K B
KB
Toh-e
A
A
Tanahashi
N
N
Tanaka
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D047992:TNF Receptor-Associated Factor 2; D014018:Tissue Distribution; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
D002352:Carrier Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D009097:Multienzyme Complexes; C101338:PSMD2 protein, human; D011506:Proteins; D012333:RNA, Messenger; C499663:RPN1 protein, S cerevisiae; D018124:Receptors, Tumor Necrosis Factor; D029701:Saccharomyces cerevisiae Proteins; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex