Limits...
Generation of high curvature membranes mediated by direct endophilin bilayer interactions.

Farsad K, Ringstad N, Takei K, Floyd SR, Rose K, De Camilli P - J. Cell Biol. (2001)

Bottom Line: This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I.Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity.Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06511, USA.

ABSTRACT
Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

Show MeSH

Related in: MedlinePlus

The endophilin B class of proteins is found on intracellular membrane compartments and also tubulates liposomes. (A) Cross-species sequence alignment (Clustal method) of the endophilin A1 and endophilin B1 proteins showing broad homology. h, human; m, mouse; lf, lamprey (L. fluviatilis); dm, D. melanogaster; ce, C. elegans. (B) Phylogenetic tree of the endophilin A1 and endophilin B1 proteins. (C) Multiple tissue Western blot using a polyclonal rabbit antibody raised against recombinant endophilin B1 showing expression primarily in heart, brain, spleen, and lung, with lower expression evident in liver and testis. Note the presence of brain-specific higher molecular weight bands. (D) Immunofluorescence of a rat brainstem frozen section using antibodies against either endophilin B1 (top) or amphiphysin 1 (bottom). Note the localization of endophilin B in a synaptic-like pattern at the cell periphery similar to the localization of amphiphysin. In addition to this synaptic staining, endophilin B localizes to intracellular particles, while amphiphysin shows a diffuse cytosolic staining pattern. (E) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in a large brainstem neuron. (F) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in CHO cells. (G) Purified recombinant endophilin B1 deforms liposomes into tubules similar to those seen with endophilin and amphiphysin. Bar, (D–F) 10 μm; (G) 100 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198845&req=5

fig5: The endophilin B class of proteins is found on intracellular membrane compartments and also tubulates liposomes. (A) Cross-species sequence alignment (Clustal method) of the endophilin A1 and endophilin B1 proteins showing broad homology. h, human; m, mouse; lf, lamprey (L. fluviatilis); dm, D. melanogaster; ce, C. elegans. (B) Phylogenetic tree of the endophilin A1 and endophilin B1 proteins. (C) Multiple tissue Western blot using a polyclonal rabbit antibody raised against recombinant endophilin B1 showing expression primarily in heart, brain, spleen, and lung, with lower expression evident in liver and testis. Note the presence of brain-specific higher molecular weight bands. (D) Immunofluorescence of a rat brainstem frozen section using antibodies against either endophilin B1 (top) or amphiphysin 1 (bottom). Note the localization of endophilin B in a synaptic-like pattern at the cell periphery similar to the localization of amphiphysin. In addition to this synaptic staining, endophilin B localizes to intracellular particles, while amphiphysin shows a diffuse cytosolic staining pattern. (E) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in a large brainstem neuron. (F) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in CHO cells. (G) Purified recombinant endophilin B1 deforms liposomes into tubules similar to those seen with endophilin and amphiphysin. Bar, (D–F) 10 μm; (G) 100 nm.

Mentions: We searched the database for other proteins which have sequence homology to the NH2 terminus of endophilin 1 to determine whether they also deform lipid bilayers. The search revealed the recently named endophilin B family of proteins (Huttner and Schmidt, 2000). This protein family shares a common domain structure with endophilin, including a COOH-terminal SH3 domain (EMBL/GenBank/DDBJ accession nos. AF263364 and AF263293) (Fig. 5 A). Antibodies were raised against the recombinant endophilin B1 protein. By Western blot analysis, this antibody recognized a 41-kD band mainly in heart, brain, spleen, and lung, with lower expression in liver and testis (Fig. 5 C). Additional higher molecular weight bands were also detected, specifically in brain, possibly representing other splice isoforms or posttranslational modifications (EMBL/GenBank/DDBJ accession no. AF263364) (Fig. 5 C). By immunofluorescence of brainstem frozen sections, endophilin B was detected in a synaptic-like pattern as shown by colocalization with synaptic markers such as amphiphysin (which also shows diffuse cytosolic staining) (Fig. 5 D). In addition, endophilin B staining was detected on intracellular membranes in the perikaryon-dendritic region of neurons (Fig. 5 D). Further analysis of this staining showed partial colocalization with the Golgi complex marker GM-130 (Fig. 5 E). In CHO cells, endophilin B localized to a perinuclear compartment strongly positive for the Golgi complex marker GM-130, in addition to a more diffuse reticular staining pattern throughout the cell (Fig. 5 F). Brefeldin A caused dispersion of the perinuclear staining by endophilin B, consistent with localization to the Golgi complex (unpublished data). There was no colocalization to endosomal compartments labeled with transferrin (unpublished data).


Generation of high curvature membranes mediated by direct endophilin bilayer interactions.

Farsad K, Ringstad N, Takei K, Floyd SR, Rose K, De Camilli P - J. Cell Biol. (2001)

The endophilin B class of proteins is found on intracellular membrane compartments and also tubulates liposomes. (A) Cross-species sequence alignment (Clustal method) of the endophilin A1 and endophilin B1 proteins showing broad homology. h, human; m, mouse; lf, lamprey (L. fluviatilis); dm, D. melanogaster; ce, C. elegans. (B) Phylogenetic tree of the endophilin A1 and endophilin B1 proteins. (C) Multiple tissue Western blot using a polyclonal rabbit antibody raised against recombinant endophilin B1 showing expression primarily in heart, brain, spleen, and lung, with lower expression evident in liver and testis. Note the presence of brain-specific higher molecular weight bands. (D) Immunofluorescence of a rat brainstem frozen section using antibodies against either endophilin B1 (top) or amphiphysin 1 (bottom). Note the localization of endophilin B in a synaptic-like pattern at the cell periphery similar to the localization of amphiphysin. In addition to this synaptic staining, endophilin B localizes to intracellular particles, while amphiphysin shows a diffuse cytosolic staining pattern. (E) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in a large brainstem neuron. (F) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in CHO cells. (G) Purified recombinant endophilin B1 deforms liposomes into tubules similar to those seen with endophilin and amphiphysin. Bar, (D–F) 10 μm; (G) 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198845&req=5

fig5: The endophilin B class of proteins is found on intracellular membrane compartments and also tubulates liposomes. (A) Cross-species sequence alignment (Clustal method) of the endophilin A1 and endophilin B1 proteins showing broad homology. h, human; m, mouse; lf, lamprey (L. fluviatilis); dm, D. melanogaster; ce, C. elegans. (B) Phylogenetic tree of the endophilin A1 and endophilin B1 proteins. (C) Multiple tissue Western blot using a polyclonal rabbit antibody raised against recombinant endophilin B1 showing expression primarily in heart, brain, spleen, and lung, with lower expression evident in liver and testis. Note the presence of brain-specific higher molecular weight bands. (D) Immunofluorescence of a rat brainstem frozen section using antibodies against either endophilin B1 (top) or amphiphysin 1 (bottom). Note the localization of endophilin B in a synaptic-like pattern at the cell periphery similar to the localization of amphiphysin. In addition to this synaptic staining, endophilin B localizes to intracellular particles, while amphiphysin shows a diffuse cytosolic staining pattern. (E) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in a large brainstem neuron. (F) Endophilin B (top) partially colocalizes with a Golgi-specific marker, GM-130 (bottom), in CHO cells. (G) Purified recombinant endophilin B1 deforms liposomes into tubules similar to those seen with endophilin and amphiphysin. Bar, (D–F) 10 μm; (G) 100 nm.
Mentions: We searched the database for other proteins which have sequence homology to the NH2 terminus of endophilin 1 to determine whether they also deform lipid bilayers. The search revealed the recently named endophilin B family of proteins (Huttner and Schmidt, 2000). This protein family shares a common domain structure with endophilin, including a COOH-terminal SH3 domain (EMBL/GenBank/DDBJ accession nos. AF263364 and AF263293) (Fig. 5 A). Antibodies were raised against the recombinant endophilin B1 protein. By Western blot analysis, this antibody recognized a 41-kD band mainly in heart, brain, spleen, and lung, with lower expression in liver and testis (Fig. 5 C). Additional higher molecular weight bands were also detected, specifically in brain, possibly representing other splice isoforms or posttranslational modifications (EMBL/GenBank/DDBJ accession no. AF263364) (Fig. 5 C). By immunofluorescence of brainstem frozen sections, endophilin B was detected in a synaptic-like pattern as shown by colocalization with synaptic markers such as amphiphysin (which also shows diffuse cytosolic staining) (Fig. 5 D). In addition, endophilin B staining was detected on intracellular membranes in the perikaryon-dendritic region of neurons (Fig. 5 D). Further analysis of this staining showed partial colocalization with the Golgi complex marker GM-130 (Fig. 5 E). In CHO cells, endophilin B localized to a perinuclear compartment strongly positive for the Golgi complex marker GM-130, in addition to a more diffuse reticular staining pattern throughout the cell (Fig. 5 F). Brefeldin A caused dispersion of the perinuclear staining by endophilin B, consistent with localization to the Golgi complex (unpublished data). There was no colocalization to endosomal compartments labeled with transferrin (unpublished data).

Bottom Line: This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I.Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity.Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06511, USA.

ABSTRACT
Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

Show MeSH
Related in: MedlinePlus