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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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Treatment of membranes with phalloidin increases the  sedimentability of p205, as well as actin. (A) Neutrophil plasma  membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients.  The initial membrane extract (Load) and gradient fractions  (lanes 1–17) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards (9S, 19S, and 30S) are indicated. (B) Phalloidin- induced shift in the sedimentability of p205. Average distribution  of p205 in gradient fractions, expressed as a percent of the total  F-actin binding at 205 kD on F-actin blot overlays (n = 3). Similar results were observed when blot strips were re-probed with  anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.
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Figure 5: Treatment of membranes with phalloidin increases the sedimentability of p205, as well as actin. (A) Neutrophil plasma membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients. The initial membrane extract (Load) and gradient fractions (lanes 1–17) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards (9S, 19S, and 30S) are indicated. (B) Phalloidin- induced shift in the sedimentability of p205. Average distribution of p205 in gradient fractions, expressed as a percent of the total F-actin binding at 205 kD on F-actin blot overlays (n = 3). Similar results were observed when blot strips were re-probed with anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.

Mentions: As expected for an F-actin–binding protein, p205 exhibited a reproducible phalloidin-induced increase in S value in TEB extracts of neutrophil plasma membranes (Fig. 5). In the presence of phalloidin, p205 sedimented as a component of a ∼30 S complex (Fig. 5, A and B) that also contained the bulk of the membrane-associated β-actin (Fig. 5 A) and γ-actin (data not shown). Although variable amounts of a ∼13 S moiety were observed in two experiments, most p205 sedimented as a ∼26 S complex in the absence of phalloidin (Fig. 5 B). Surprisingly, whereas much of the actin was rendered monomeric by the harsh buffer conditions, significant amounts continued to sediment with high S values in the absence of phalloidin stabilization (Fig. 5 A). In contrast to the behavior of p205 and actin, little or no fodrin, myosin, ezrin, or moesin exhibited significant phalloidin-induced shifts in sedimentability under these conditions (Fig. 5 A). Thus, p205 apparently forms large complexes with endogenous actin under conditions that suggest an extremely tight association, direct or indirect, in the neutrophil membrane skeleton.


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)

Treatment of membranes with phalloidin increases the  sedimentability of p205, as well as actin. (A) Neutrophil plasma  membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients.  The initial membrane extract (Load) and gradient fractions  (lanes 1–17) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards (9S, 19S, and 30S) are indicated. (B) Phalloidin- induced shift in the sedimentability of p205. Average distribution  of p205 in gradient fractions, expressed as a percent of the total  F-actin binding at 205 kD on F-actin blot overlays (n = 3). Similar results were observed when blot strips were re-probed with  anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140202&req=5

Figure 5: Treatment of membranes with phalloidin increases the sedimentability of p205, as well as actin. (A) Neutrophil plasma membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients. The initial membrane extract (Load) and gradient fractions (lanes 1–17) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards (9S, 19S, and 30S) are indicated. (B) Phalloidin- induced shift in the sedimentability of p205. Average distribution of p205 in gradient fractions, expressed as a percent of the total F-actin binding at 205 kD on F-actin blot overlays (n = 3). Similar results were observed when blot strips were re-probed with anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.
Mentions: As expected for an F-actin–binding protein, p205 exhibited a reproducible phalloidin-induced increase in S value in TEB extracts of neutrophil plasma membranes (Fig. 5). In the presence of phalloidin, p205 sedimented as a component of a ∼30 S complex (Fig. 5, A and B) that also contained the bulk of the membrane-associated β-actin (Fig. 5 A) and γ-actin (data not shown). Although variable amounts of a ∼13 S moiety were observed in two experiments, most p205 sedimented as a ∼26 S complex in the absence of phalloidin (Fig. 5 B). Surprisingly, whereas much of the actin was rendered monomeric by the harsh buffer conditions, significant amounts continued to sediment with high S values in the absence of phalloidin stabilization (Fig. 5 A). In contrast to the behavior of p205 and actin, little or no fodrin, myosin, ezrin, or moesin exhibited significant phalloidin-induced shifts in sedimentability under these conditions (Fig. 5 A). Thus, p205 apparently forms large complexes with endogenous actin under conditions that suggest an extremely tight association, direct or indirect, in the neutrophil membrane skeleton.

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