Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.
Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.
Zheng
Chun-Lei
CL
Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
Sumizawa
Tomoyuki
T
Che
Xiao-Fang
XF
Tsuyama
Shinichiro
S
Furukawa
Tatsuhiko
T
Haraguchi
Misako
M
Gao
Hui
H
Gotanda
Takenari
T
Jueng
Hei-Cheul
HC
Murata
Fusayoshi
F
Akiyama
Shin-Ichi
S
D001665:Binding Sites; D002292:Carcinoma, Renal Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000378:metabolism*; D008565:Membrane Proteins; D009124:Muscle Proteins; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D020394:Vault Ribonucleoprotein Particles / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000099187:Major Vault Protein
D008565:Membrane Proteins; D009124:Muscle Proteins; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C116148:STBD1 protein, human; C449359:vault poly(ADP-ribose) polymerase