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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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p193 exists in cells associated with vaults and in other subcellular fractions. Comparison of levels of p193 during biochemical fractionation of HeLa cells. (A) Cellular protein fractions, nuclear (N), supernatant S100 (S), and microsomal pellet P100 (P) were generated from HeLa extracts and resolved by SDS-PAGE (see Materials and Methods). Immunoblot analysis was carried out using affinity-purified polyclonal antibody prepared against recombinant p193 fragment. (B) The P100 fraction was further fractionated on a discontinuous sucrose gradient (20/30/40/45/50/60%), and the proteins from the gradient fractions were resolved by SDS-PAGE. Immunoblot analysis was carried out using either affinity-purified p193 polyclonal antibody (top) or affinity-purified anti-vault polyclonal antibody (bottom). Sucrose gradient fraction sources are indicated at the top. P100 represents the starting material, and the vault sample was purified from rat liver (a lighter exposure of the detected MVP in purified vaults is shown). Arrows indicate the positions of p193 and MVP.
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Figure 7: p193 exists in cells associated with vaults and in other subcellular fractions. Comparison of levels of p193 during biochemical fractionation of HeLa cells. (A) Cellular protein fractions, nuclear (N), supernatant S100 (S), and microsomal pellet P100 (P) were generated from HeLa extracts and resolved by SDS-PAGE (see Materials and Methods). Immunoblot analysis was carried out using affinity-purified polyclonal antibody prepared against recombinant p193 fragment. (B) The P100 fraction was further fractionated on a discontinuous sucrose gradient (20/30/40/45/50/60%), and the proteins from the gradient fractions were resolved by SDS-PAGE. Immunoblot analysis was carried out using either affinity-purified p193 polyclonal antibody (top) or affinity-purified anti-vault polyclonal antibody (bottom). Sucrose gradient fraction sources are indicated at the top. P100 represents the starting material, and the vault sample was purified from rat liver (a lighter exposure of the detected MVP in purified vaults is shown). Arrows indicate the positions of p193 and MVP.

Mentions: A polyclonal anti-p193 antibody was generated from bacterially expressed fragments of p193 (aa 408–611 and 1471–1724; see Materials and Methods). The anti-p193 antibody recognizes a single protein species of 193 kD by immunoblot analysis (Fig. 7 A). To compare the subcellular distribution of p193 with MVP, extracts from tissue culture cells were isolated and fractionated on a discontinuous sucrose gradient followed by immunoblot analysis (Fig. 7). Vaults are cytoplasmic particles that typically pellet with the microsomes at 100,000 g (Kedersha and Rome 1986). Detergent-lysed HeLa cells were centrifuged at 20,000 g, resulting in a nuclear (N) pellet. The supernatant was further fractionated by centrifugation at 100,000 g and the supernatant (S100) and pellet (P100) fractions were analyzed by immunoblotting with anti-p193 antibody. Interestingly, unlike MVP, which primarily fractionates with the P100, all of the fractions contained the p193 protein (Fig. 7 A). We should note that the N fraction does not represent purified nuclei, and a certain portion of the cells are in mitosis at any given time, so the amount of p193 detected by immunoblotting may not be comparable to that seen by immunofluorescence (see below). Further fractionation of the P100 extract on a discontinuous sucrose gradient revealed that the majority of p193 sedimented to the 45/50% sucrose layer, coinciding with the pattern observed for the MVP (Fig. 7 B). These results suggest that all of the p193 protein in the P100 fraction is associated with the vault particle. Immunoblot analysis of vaults purified from rat liver revealed that the p193 vault protein is recognized by the anti-p193 antibody, further confirming its association with vaults (Fig. 7 B). The shifted mobility of p193 in vaults purified from rat liver is probably due to species-specific differences.


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)

p193 exists in cells associated with vaults and in other subcellular fractions. Comparison of levels of p193 during biochemical fractionation of HeLa cells. (A) Cellular protein fractions, nuclear (N), supernatant S100 (S), and microsomal pellet P100 (P) were generated from HeLa extracts and resolved by SDS-PAGE (see Materials and Methods). Immunoblot analysis was carried out using affinity-purified polyclonal antibody prepared against recombinant p193 fragment. (B) The P100 fraction was further fractionated on a discontinuous sucrose gradient (20/30/40/45/50/60%), and the proteins from the gradient fractions were resolved by SDS-PAGE. Immunoblot analysis was carried out using either affinity-purified p193 polyclonal antibody (top) or affinity-purified anti-vault polyclonal antibody (bottom). Sucrose gradient fraction sources are indicated at the top. P100 represents the starting material, and the vault sample was purified from rat liver (a lighter exposure of the detected MVP in purified vaults is shown). Arrows indicate the positions of p193 and MVP.
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Figure 7: p193 exists in cells associated with vaults and in other subcellular fractions. Comparison of levels of p193 during biochemical fractionation of HeLa cells. (A) Cellular protein fractions, nuclear (N), supernatant S100 (S), and microsomal pellet P100 (P) were generated from HeLa extracts and resolved by SDS-PAGE (see Materials and Methods). Immunoblot analysis was carried out using affinity-purified polyclonal antibody prepared against recombinant p193 fragment. (B) The P100 fraction was further fractionated on a discontinuous sucrose gradient (20/30/40/45/50/60%), and the proteins from the gradient fractions were resolved by SDS-PAGE. Immunoblot analysis was carried out using either affinity-purified p193 polyclonal antibody (top) or affinity-purified anti-vault polyclonal antibody (bottom). Sucrose gradient fraction sources are indicated at the top. P100 represents the starting material, and the vault sample was purified from rat liver (a lighter exposure of the detected MVP in purified vaults is shown). Arrows indicate the positions of p193 and MVP.
Mentions: A polyclonal anti-p193 antibody was generated from bacterially expressed fragments of p193 (aa 408–611 and 1471–1724; see Materials and Methods). The anti-p193 antibody recognizes a single protein species of 193 kD by immunoblot analysis (Fig. 7 A). To compare the subcellular distribution of p193 with MVP, extracts from tissue culture cells were isolated and fractionated on a discontinuous sucrose gradient followed by immunoblot analysis (Fig. 7). Vaults are cytoplasmic particles that typically pellet with the microsomes at 100,000 g (Kedersha and Rome 1986). Detergent-lysed HeLa cells were centrifuged at 20,000 g, resulting in a nuclear (N) pellet. The supernatant was further fractionated by centrifugation at 100,000 g and the supernatant (S100) and pellet (P100) fractions were analyzed by immunoblotting with anti-p193 antibody. Interestingly, unlike MVP, which primarily fractionates with the P100, all of the fractions contained the p193 protein (Fig. 7 A). We should note that the N fraction does not represent purified nuclei, and a certain portion of the cells are in mitosis at any given time, so the amount of p193 detected by immunoblotting may not be comparable to that seen by immunofluorescence (see below). Further fractionation of the P100 extract on a discontinuous sucrose gradient revealed that the majority of p193 sedimented to the 45/50% sucrose layer, coinciding with the pattern observed for the MVP (Fig. 7 B). These results suggest that all of the p193 protein in the P100 fraction is associated with the vault particle. Immunoblot analysis of vaults purified from rat liver revealed that the p193 vault protein is recognized by the anti-p193 antibody, further confirming its association with vaults (Fig. 7 B). The shifted mobility of p193 in vaults purified from rat liver is probably due to species-specific differences.

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