Figure 3. The ALPS motif binds highly... | Rockefeller University Press

Figure 3.

$Figure 3. The +ALPS motif binds highly curved membranes with anionic phospholipid. (A) GST-tagged Gcs1(186–352) (0.5 mM) was incubated with Golgi-mix liposomes (0.5-mM lipids) of decreasing size produced by extrusion through filter pores of the size indicated. After centrifugation in a density gradient to float liposomes, the top fractions were collected and analyzed by SDS-PAGE and Coomassie staining. Gcs1(186–352) in the top fraction from a liposome-free control was subtracted from the band intensities shown for quantitation. Error bars show variation observed between three independent experiments using different batches of extruded liposomes. (B) GST-tagged Gcs1(186–285) and Gcs1(186–285)K210Q were incubated with 200 nm DOPC liposomes with increasing percentages of DOPS. After centrifugation, the top fractions were collected and quantified by Western blotting using anti-GST antibody. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars indicate mean + SD. (C) Binding of GST-Gcs1(186–285) to 100 nm DOPC liposomes with or without the indicated anionic phospholipids. Data are expressed as the percentage of input protein. The data shown are from a single representative experiment out of two repeats.$

The +ALPS motif binds highly curved membranes with anionic phospholipid. (A) GST-tagged Gcs1(186–352) (0.5 mM) was incubated with Golgi-mix liposomes (0.5-mM lipids) of decreasing size produced by extrusion through filter pores of the size indicated. After centrifugation in a density gradient to float liposomes, the top fractions were collected and analyzed by SDS-PAGE and Coomassie staining. Gcs1(186–352) in the top fraction from a liposome-free control was subtracted from the band intensities shown for quantitation. Error bars show variation observed between three independent experiments using different batches of extruded liposomes. (B) GST-tagged Gcs1(186–285) and Gcs1(186–285)^K210Q were incubated with 200 nm DOPC liposomes with increasing percentages of DOPS. After centrifugation, the top fractions were collected and quantified by Western blotting using anti-GST antibody. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars indicate mean + SD. (C) Binding of GST-Gcs1(186–285) to 100 nm DOPC liposomes with or without the indicated anionic phospholipids. Data are expressed as the percentage of input protein. The data shown are from a single representative experiment out of two repeats.