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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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Neutrophil plasma  membranes are highly enriched in p205. Fractions  highly enriched in plasma  membranes (A) or secretory  vesicles (B) were analyzed for the presence of p205 by F-actin  blot overlay (C). Aliquots (100 μg protein) were fractionated on  a 5% SDS–polyacrylamide gel, transferred to nitrocellulose, and  then probed with 125I-labeled F-actin. Lane 1, plasma membranes; lane 2, secretory vesicles; and lane 3, cytosol. E/M, the  position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3).
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Figure 1: Neutrophil plasma membranes are highly enriched in p205. Fractions highly enriched in plasma membranes (A) or secretory vesicles (B) were analyzed for the presence of p205 by F-actin blot overlay (C). Aliquots (100 μg protein) were fractionated on a 5% SDS–polyacrylamide gel, transferred to nitrocellulose, and then probed with 125I-labeled F-actin. Lane 1, plasma membranes; lane 2, secretory vesicles; and lane 3, cytosol. E/M, the position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3).

Mentions: As detected by blot overlays with 125I-labeled F-actin (17), moesin, ezrin, and a polypeptide with an Mr of 205,000 (p205) were previously described as major actin-binding proteins in a bovine neutrophil membrane fraction called the γ fraction (66). The γ fraction contains both large sheets of plasma membrane and “secretory vesicles,” which are intracellular vesicles of similar density and composition that are believed to represent mobilizable intracellular stores of plasma membrane proteins involved in cell adhesion and other activation-associated surface processes (9). Taking advantage of a recently described technique for separating human neutrophil membranes containing latent vs. surface-exposed alkaline phosphatase (21), we found that the use of a modified Percoll gradient does indeed separate the γ fraction into two membrane populations (Fig. 1). The less dense fraction (density <1.04) is enriched in large membrane sheets with associated amorphous filamentous structures (Fig. 1 A) and corresponds to the peak of surface-exposed alkaline phosphatase (not shown). Thus, the less dense fraction appears to be mostly plasma membrane. The denser fraction (density between 1.04 and 1.06) corresponds to the peak of latent alkaline phosphatase (not shown) and contains predominantly small osmophilic vesicles (Fig. 1 B). These vesicles probably represent secretory vesicles although some mitochondria are also observed in this fraction. While large amounts of all three major F-actin–binding proteins (p205, ezrin, moesin) were found in the plasma membrane fraction (Fig. 1 C, lane 1), the enrichment was greatest for p205 since ezrin and moesin also were present in the secretory vesicle (Fig. 1 C, lane 2) and cytosolic (Fig. 1 C, lane 3) fractions. As reported previously (66), no F-actin–binding proteins were observed in the pooled granule fraction. There was at least 20-fold more p205 in the plasma membrane fraction than in the secretory vesicle fraction, and 10–15-fold more than in cytosol. This large enrichment suggests an intimate association of p205 with 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)

Neutrophil plasma  membranes are highly enriched in p205. Fractions  highly enriched in plasma  membranes (A) or secretory  vesicles (B) were analyzed for the presence of p205 by F-actin  blot overlay (C). Aliquots (100 μg protein) were fractionated on  a 5% SDS–polyacrylamide gel, transferred to nitrocellulose, and  then probed with 125I-labeled F-actin. Lane 1, plasma membranes; lane 2, secretory vesicles; and lane 3, cytosol. E/M, the  position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140202&req=5

Figure 1: Neutrophil plasma membranes are highly enriched in p205. Fractions highly enriched in plasma membranes (A) or secretory vesicles (B) were analyzed for the presence of p205 by F-actin blot overlay (C). Aliquots (100 μg protein) were fractionated on a 5% SDS–polyacrylamide gel, transferred to nitrocellulose, and then probed with 125I-labeled F-actin. Lane 1, plasma membranes; lane 2, secretory vesicles; and lane 3, cytosol. E/M, the position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3).
Mentions: As detected by blot overlays with 125I-labeled F-actin (17), moesin, ezrin, and a polypeptide with an Mr of 205,000 (p205) were previously described as major actin-binding proteins in a bovine neutrophil membrane fraction called the γ fraction (66). The γ fraction contains both large sheets of plasma membrane and “secretory vesicles,” which are intracellular vesicles of similar density and composition that are believed to represent mobilizable intracellular stores of plasma membrane proteins involved in cell adhesion and other activation-associated surface processes (9). Taking advantage of a recently described technique for separating human neutrophil membranes containing latent vs. surface-exposed alkaline phosphatase (21), we found that the use of a modified Percoll gradient does indeed separate the γ fraction into two membrane populations (Fig. 1). The less dense fraction (density <1.04) is enriched in large membrane sheets with associated amorphous filamentous structures (Fig. 1 A) and corresponds to the peak of surface-exposed alkaline phosphatase (not shown). Thus, the less dense fraction appears to be mostly plasma membrane. The denser fraction (density between 1.04 and 1.06) corresponds to the peak of latent alkaline phosphatase (not shown) and contains predominantly small osmophilic vesicles (Fig. 1 B). These vesicles probably represent secretory vesicles although some mitochondria are also observed in this fraction. While large amounts of all three major F-actin–binding proteins (p205, ezrin, moesin) were found in the plasma membrane fraction (Fig. 1 C, lane 1), the enrichment was greatest for p205 since ezrin and moesin also were present in the secretory vesicle (Fig. 1 C, lane 2) and cytosolic (Fig. 1 C, lane 3) fractions. As reported previously (66), no F-actin–binding proteins were observed in the pooled granule fraction. There was at least 20-fold more p205 in the plasma membrane fraction than in the secretory vesicle fraction, and 10–15-fold more than in cytosol. This large enrichment suggests an intimate association of p205 with 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