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Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

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p205 is a peripheral component of the neutrophil  plasma membrane skeleton. (A) Neutrophil plasma membranes  were extracted with either buffer alone (lane 1), or buffer containing a final concentration of 2.5 mM MgATP (lane 2), 0.25 M  KCl (lane 3), 1.0 M KCl (lane 4), 0.1 M sodium carbonate (lane 5),  or 0.1 M NaOH (lane 6). For each extraction condition, high  speed supernatants (S) and pellets (P) from 130-μg membranes  were electrophoresed, blotted, and then probed with 125I-labeled  F-actin and with specific antibodies. (B) Neutrophil plasma  membranes (100 μg per treatment) were extracted with either  buffer alone (lane 1), or buffer containing 1% Triton X-100 (lane  2), 1% Triton X-100, 50 mM NaCl (lane 3), 1% Triton X-100, 250  mM NaCl (lane 4), 3% octylglucoside, 250 mM NaCl (lane 5), or  0.1% SDS (lane 6) and processed as above. A positive control  consisted of membranes extracted with 1% SDS at 70°C for 10  min (lane 7). The higher mobility F-actin–binding polypeptide  present in B is consistently observed after detergent treatment;  this band also reacts with an antibody against p205 sequences  (see below), suggesting a close structural relationship with p205.
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Figure 2: p205 is a peripheral component of the neutrophil plasma membrane skeleton. (A) Neutrophil plasma membranes were extracted with either buffer alone (lane 1), or buffer containing a final concentration of 2.5 mM MgATP (lane 2), 0.25 M KCl (lane 3), 1.0 M KCl (lane 4), 0.1 M sodium carbonate (lane 5), or 0.1 M NaOH (lane 6). For each extraction condition, high speed supernatants (S) and pellets (P) from 130-μg membranes were electrophoresed, blotted, and then probed with 125I-labeled F-actin and with specific antibodies. (B) Neutrophil plasma membranes (100 μg per treatment) were extracted with either buffer alone (lane 1), or buffer containing 1% Triton X-100 (lane 2), 1% Triton X-100, 50 mM NaCl (lane 3), 1% Triton X-100, 250 mM NaCl (lane 4), 3% octylglucoside, 250 mM NaCl (lane 5), or 0.1% SDS (lane 6) and processed as above. A positive control consisted of membranes extracted with 1% SDS at 70°C for 10 min (lane 7). The higher mobility F-actin–binding polypeptide present in B is consistently observed after detergent treatment; this band also reacts with an antibody against p205 sequences (see below), suggesting a close structural relationship with p205.

Mentions: To explore the nature of the interaction between p205 and the plasma membrane, we extracted purified neutrophil plasma membranes with a series of salt and detergent solutions (Fig. 2). We found that buffers containing 2.5 mM MgATP, a reagent that extracts most of the similarly sized myosin II, had no effect on the membrane association of p205 (Fig. 2 A, lanes 2S and 2P). Similarly, moderately high salt concentrations (0.25 M) that extract significant amounts of membrane-bound moesin and ezrin, had no detectable effect on the extractability of p205 (Fig. 2 A, lanes 3S and 3P). Even sodium carbonate, a reagent that extracts many peripherally bound proteins (44), had no effect on the membrane association of p205 (Fig. 2 A, lanes 5S and 5P). On the other hand, p205 was partially extracted at salt concentrations >1 M (Fig. 2 A, lanes 4S and 4P) and was almost completely extracted by 0.1 M NaOH (Fig. 2 A, lanes 6S and 6P), indicating that p205 is a tightly bound peripheral protein, and not an integral component, of the plasma membrane.


Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

p205 is a peripheral component of the neutrophil  plasma membrane skeleton. (A) Neutrophil plasma membranes  were extracted with either buffer alone (lane 1), or buffer containing a final concentration of 2.5 mM MgATP (lane 2), 0.25 M  KCl (lane 3), 1.0 M KCl (lane 4), 0.1 M sodium carbonate (lane 5),  or 0.1 M NaOH (lane 6). For each extraction condition, high  speed supernatants (S) and pellets (P) from 130-μg membranes  were electrophoresed, blotted, and then probed with 125I-labeled  F-actin and with specific antibodies. (B) Neutrophil plasma  membranes (100 μg per treatment) were extracted with either  buffer alone (lane 1), or buffer containing 1% Triton X-100 (lane  2), 1% Triton X-100, 50 mM NaCl (lane 3), 1% Triton X-100, 250  mM NaCl (lane 4), 3% octylglucoside, 250 mM NaCl (lane 5), or  0.1% SDS (lane 6) and processed as above. A positive control  consisted of membranes extracted with 1% SDS at 70°C for 10  min (lane 7). The higher mobility F-actin–binding polypeptide  present in B is consistently observed after detergent treatment;  this band also reacts with an antibody against p205 sequences  (see below), suggesting a close structural relationship with p205.
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Related In: Results  -  Collection

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Figure 2: p205 is a peripheral component of the neutrophil plasma membrane skeleton. (A) Neutrophil plasma membranes were extracted with either buffer alone (lane 1), or buffer containing a final concentration of 2.5 mM MgATP (lane 2), 0.25 M KCl (lane 3), 1.0 M KCl (lane 4), 0.1 M sodium carbonate (lane 5), or 0.1 M NaOH (lane 6). For each extraction condition, high speed supernatants (S) and pellets (P) from 130-μg membranes were electrophoresed, blotted, and then probed with 125I-labeled F-actin and with specific antibodies. (B) Neutrophil plasma membranes (100 μg per treatment) were extracted with either buffer alone (lane 1), or buffer containing 1% Triton X-100 (lane 2), 1% Triton X-100, 50 mM NaCl (lane 3), 1% Triton X-100, 250 mM NaCl (lane 4), 3% octylglucoside, 250 mM NaCl (lane 5), or 0.1% SDS (lane 6) and processed as above. A positive control consisted of membranes extracted with 1% SDS at 70°C for 10 min (lane 7). The higher mobility F-actin–binding polypeptide present in B is consistently observed after detergent treatment; this band also reacts with an antibody against p205 sequences (see below), suggesting a close structural relationship with p205.
Mentions: To explore the nature of the interaction between p205 and the plasma membrane, we extracted purified neutrophil plasma membranes with a series of salt and detergent solutions (Fig. 2). We found that buffers containing 2.5 mM MgATP, a reagent that extracts most of the similarly sized myosin II, had no effect on the membrane association of p205 (Fig. 2 A, lanes 2S and 2P). Similarly, moderately high salt concentrations (0.25 M) that extract significant amounts of membrane-bound moesin and ezrin, had no detectable effect on the extractability of p205 (Fig. 2 A, lanes 3S and 3P). Even sodium carbonate, a reagent that extracts many peripherally bound proteins (44), had no effect on the membrane association of p205 (Fig. 2 A, lanes 5S and 5P). On the other hand, p205 was partially extracted at salt concentrations >1 M (Fig. 2 A, lanes 4S and 4P) and was almost completely extracted by 0.1 M NaOH (Fig. 2 A, lanes 6S and 6P), indicating that p205 is a tightly bound peripheral protein, and not an integral component, of the plasma membrane.

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

Show MeSH
Related in: MedlinePlus