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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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Purification of p205 by SDS-PAGE. (A) Neutrophil  plasma membranes (lane 1) and Triton X-100–insoluble pellets  (lanes 2–5) were separated on a 5% polyacrylamide gel and  stained with silver (lanes 1 and 2), or electrotransfered to nitrocellulose and probed with either 125I-labeled F-actin (lane 3), or  with antibodies against myosin II (lane 4), or nonerythroid spectrin/fodrin (lane 5). Loads represent 100 μg membranes or equivalent amounts of Triton X-100–insoluble pellets. The location of  p205 is indicated (arrowheads). (B) Eight microsequences were  obtained from proteolytic digests of SDS-PAGE–purified p205.  Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences (pepA and pepB).  Residues at variance with the deduced amino acid sequence (Fig.  11 A) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.
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Figure 3: Purification of p205 by SDS-PAGE. (A) Neutrophil plasma membranes (lane 1) and Triton X-100–insoluble pellets (lanes 2–5) were separated on a 5% polyacrylamide gel and stained with silver (lanes 1 and 2), or electrotransfered to nitrocellulose and probed with either 125I-labeled F-actin (lane 3), or with antibodies against myosin II (lane 4), or nonerythroid spectrin/fodrin (lane 5). Loads represent 100 μg membranes or equivalent amounts of Triton X-100–insoluble pellets. The location of p205 is indicated (arrowheads). (B) Eight microsequences were obtained from proteolytic digests of SDS-PAGE–purified p205. Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences (pepA and pepB). Residues at variance with the deduced amino acid sequence (Fig. 11 A) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.

Mentions: The inextractability of p205 under conditions that solubilized most membrane and membrane skeleton proteins, in conjunction with the large amounts of plasma membrane obtainable from bovine neutrophils, suggested that this protein should be readily purified by differential extraction followed by preparative SDS-PAGE. We thus extracted plasma membranes with a buffer containing 1% Triton X-100, 0.25 M NaCl, and 1 mM MgATP to identify proteins that are tightly associated with the neutrophil plasma membrane skeleton (Fig. 3 A, lane 2 vs. lane 1). The inclusion of MgATP in this buffer resulted in the removal of most of the myosin II that migrated near p205 on SDS gels. When run on a long, 5% polyacrylamide gel, there was a clear separation of p205 from residual myosin and other similarly sized membrane skeleton proteins (Fig. 3 A, lanes 2–5). Microsequencing of the band corresponding to p205 generated a total of eight peptide sequences (Fig. 3 B). None of these peptide sequences were significantly similar to any protein sequence in the nonredundant, updated protein databases, indicating that p205 is a previously uncharacterized protein.


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)

Purification of p205 by SDS-PAGE. (A) Neutrophil  plasma membranes (lane 1) and Triton X-100–insoluble pellets  (lanes 2–5) were separated on a 5% polyacrylamide gel and  stained with silver (lanes 1 and 2), or electrotransfered to nitrocellulose and probed with either 125I-labeled F-actin (lane 3), or  with antibodies against myosin II (lane 4), or nonerythroid spectrin/fodrin (lane 5). Loads represent 100 μg membranes or equivalent amounts of Triton X-100–insoluble pellets. The location of  p205 is indicated (arrowheads). (B) Eight microsequences were  obtained from proteolytic digests of SDS-PAGE–purified p205.  Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences (pepA and pepB).  Residues at variance with the deduced amino acid sequence (Fig.  11 A) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140202&req=5

Figure 3: Purification of p205 by SDS-PAGE. (A) Neutrophil plasma membranes (lane 1) and Triton X-100–insoluble pellets (lanes 2–5) were separated on a 5% polyacrylamide gel and stained with silver (lanes 1 and 2), or electrotransfered to nitrocellulose and probed with either 125I-labeled F-actin (lane 3), or with antibodies against myosin II (lane 4), or nonerythroid spectrin/fodrin (lane 5). Loads represent 100 μg membranes or equivalent amounts of Triton X-100–insoluble pellets. The location of p205 is indicated (arrowheads). (B) Eight microsequences were obtained from proteolytic digests of SDS-PAGE–purified p205. Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences (pepA and pepB). Residues at variance with the deduced amino acid sequence (Fig. 11 A) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.
Mentions: The inextractability of p205 under conditions that solubilized most membrane and membrane skeleton proteins, in conjunction with the large amounts of plasma membrane obtainable from bovine neutrophils, suggested that this protein should be readily purified by differential extraction followed by preparative SDS-PAGE. We thus extracted plasma membranes with a buffer containing 1% Triton X-100, 0.25 M NaCl, and 1 mM MgATP to identify proteins that are tightly associated with the neutrophil plasma membrane skeleton (Fig. 3 A, lane 2 vs. lane 1). The inclusion of MgATP in this buffer resulted in the removal of most of the myosin II that migrated near p205 on SDS gels. When run on a long, 5% polyacrylamide gel, there was a clear separation of p205 from residual myosin and other similarly sized membrane skeleton proteins (Fig. 3 A, lanes 2–5). Microsequencing of the band corresponding to p205 generated a total of eight peptide sequences (Fig. 3 B). None of these peptide sequences were significantly similar to any protein sequence in the nonredundant, updated protein databases, indicating that p205 is a previously uncharacterized protein.

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