Figure 3. DysF is a PA-binding domain.... | Rockefeller University Press

Figure 3.

$DysF is a PA-binding domain. (A) DysF domain from Pex30 binds to PA and more weakly to phosphoinositides. Purified DysF was incubated with the indicated lipids immobilized in a nitrocellulose membrane. Pex30 DysF was expressed as a fusion protein to the epitope tags indicated and was detected with anti-HA antibody. IB, immunoblot. (B) DysF domain from Pex30 binds to PA-containing liposomes. Left: purified DysF and liposomes of defined concentration were pre-mixed for 30 min, layered by sucrose solution, and subjected to centrifugation for liposome flotation, as depicted. The sucrose gradient was fractionated into four fractions, and the samples were subjected to SDS-PAGE and proteins were stained with instant blue. Right: quantification of the percentage of DysF cofractionating with liposomes to the top fraction of experiments shown in C. The bars represent the SD. (C) Liposome flotation assay of His-Sumo-Pex30-DysF as described in B using liposomes containing different PA concentrations or the indicated concentration of PS, PI, or PI(4)P. Quantification of the percentage of the protein interacting with the liposomes is represented in B. The position of molecular weight markers (in kDa) is indicated. Source data are available for this figure: SourceData F3.$

DysF is a PA-binding domain. (A) DysF domain from Pex30 binds to PA and more weakly to phosphoinositides. Purified DysF was incubated with the indicated lipids immobilized in a nitrocellulose membrane. Pex30 DysF was expressed as a fusion protein to the epitope tags indicated and was detected with anti-HA antibody. IB, immunoblot. (B) DysF domain from Pex30 binds to PA-containing liposomes. Left: purified DysF and liposomes of defined concentration were pre-mixed for 30 min, layered by sucrose solution, and subjected to centrifugation for liposome flotation, as depicted. The sucrose gradient was fractionated into four fractions, and the samples were subjected to SDS-PAGE and proteins were stained with instant blue. Right: quantification of the percentage of DysF cofractionating with liposomes to the top fraction of experiments shown in C. The bars represent the SD. (C) Liposome flotation assay of His-Sumo-Pex30-DysF as described in B using liposomes containing different PA concentrations or the indicated concentration of PS, PI, or PI(4)P. Quantification of the percentage of the protein interacting with the liposomes is represented in B. The position of molecular weight markers (in kDa) is indicated. Source data are available for this figure: SourceData F3.

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