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The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH - J. Cell Biol. (1999)

Bottom Line: Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP.Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s).A portion of p193 is nuclear and localizes to the mitotic spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA. vkick@medne.tucla.edu

ABSTRACT
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

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In purified vaults, p193 is a PARP that ADP-ribosylates itself and the MVP. Vaults (3 μg) purified from rat liver were incubated in the presence of [32P]NAD+ (as indicated) and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel), and by autoradiography (right panel). Reactions were supplemented with either unlabeled NAD+ (Cold Chase) or with the PARP inhibitor 3ABA (Inhibitor). Arrows indicate the specific ADP-ribosylated proteins. The bracket indicates the smear of higher molecular weight ADP-ribosylated products.
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Figure 5: In purified vaults, p193 is a PARP that ADP-ribosylates itself and the MVP. Vaults (3 μg) purified from rat liver were incubated in the presence of [32P]NAD+ (as indicated) and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel), and by autoradiography (right panel). Reactions were supplemented with either unlabeled NAD+ (Cold Chase) or with the PARP inhibitor 3ABA (Inhibitor). Arrows indicate the specific ADP-ribosylated proteins. The bracket indicates the smear of higher molecular weight ADP-ribosylated products.

Mentions: Next, we wanted to investigate whether full-length endogenous p193 within the vault particle would possess enzymatic activity. Highly purified vault particles were incubated with [32P]NAD+ in the presence and absence of inhibitor or unlabeled NAD+. The most prominently modified protein in purified vaults was the MVP. However, there was some labeling in the vicinity of the p193 and a high molecular weight smear was also detected (Fig. 5). Modification of all of these products was competed for by the addition of unlabeled NAD+ and partially competed by the addition of the inhibitor 3ABA. These data indicate that full-length p193 is a PARP that is active in the vault particle with at least one specific substrate, MVP.


The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH - J. Cell Biol. (1999)

In purified vaults, p193 is a PARP that ADP-ribosylates itself and the MVP. Vaults (3 μg) purified from rat liver were incubated in the presence of [32P]NAD+ (as indicated) and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel), and by autoradiography (right panel). Reactions were supplemented with either unlabeled NAD+ (Cold Chase) or with the PARP inhibitor 3ABA (Inhibitor). Arrows indicate the specific ADP-ribosylated proteins. The bracket indicates the smear of higher molecular weight ADP-ribosylated products.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169495&req=5

Figure 5: In purified vaults, p193 is a PARP that ADP-ribosylates itself and the MVP. Vaults (3 μg) purified from rat liver were incubated in the presence of [32P]NAD+ (as indicated) and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel), and by autoradiography (right panel). Reactions were supplemented with either unlabeled NAD+ (Cold Chase) or with the PARP inhibitor 3ABA (Inhibitor). Arrows indicate the specific ADP-ribosylated proteins. The bracket indicates the smear of higher molecular weight ADP-ribosylated products.
Mentions: Next, we wanted to investigate whether full-length endogenous p193 within the vault particle would possess enzymatic activity. Highly purified vault particles were incubated with [32P]NAD+ in the presence and absence of inhibitor or unlabeled NAD+. The most prominently modified protein in purified vaults was the MVP. However, there was some labeling in the vicinity of the p193 and a high molecular weight smear was also detected (Fig. 5). Modification of all of these products was competed for by the addition of unlabeled NAD+ and partially competed by the addition of the inhibitor 3ABA. These data indicate that full-length p193 is a PARP that is active in the vault particle with at least one specific substrate, MVP.

Bottom Line: Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP.Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s).A portion of p193 is nuclear and localizes to the mitotic spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA. vkick@medne.tucla.edu

ABSTRACT
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

Show MeSH
Related in: MedlinePlus