Arfaptin2 induces membrane tubulation at high concentration.

<p>Golgi-mix GUVs containing 1% of the red fluorescent lipid BodTRCer were incubated with (A) 3 µM Arfaptin2-Alexa⁴⁸⁸ and 3 µM Arf1:GDP or (B) 3 µM Arfaptin2-Alexa⁴⁸⁸ and 3 µM Arf1:GTPγS. Scale bar: 10 µm.</p

The binding of Arfaptin2 to liposomes requires Arf1 activation and increases with membrane curvature.

<p>A. Flotation assay. Arfaptin2 and Arf1:GDP (left) or Arf1:GTP (right) (0.75 µM) were co-incubated with Golgi-mix liposomes (0.75 mM lipids) extruded through filters with pores of decreasing sizes (200 nm, 100 nm, 50 nm and 30 nm). The suspension was adjusted to 30% w/v sucrose and overlaid with two cushions of decreasing sucrose density. After centrifugation, the top (liposomes) and bottom (unbound proteins) fractions were collected and analysed by SDS-PAGE. Direct Alexa⁴⁸⁸ fluorescence (top panels) was observed before Sypro-orange staining (bottom panels). B. Protein quantification from the Sypro-orange stained gels shown in A. The black bars correspond to Arfaptin2 and the white/black striped bars to Arf1. C. FRET assay. Arfaptin2-Alexa⁴⁸⁸ (0.5 µM) and myr-Arf1-H6 were mixed with Golgi-mix liposomes of various sizes containing 1 mol% TRITC-DHPE. At the indicated times, GTP (0.1 mM), EDTA (2 mM), MgCl2 (2 mM) and ArfGAP1 (10 nM) were added. Alexa⁴⁸⁸ fluorescence was followed in real time. Arfaptin2 was recruited to the membranes after Arf1 activation (GTP). Arfaptin2 recruitment increased as a function of membrane curvature.</p

Arfaptin2 binds preferentially to membrane tubes connected to GUVs when Arf1 is in its active GTP-bound state.

<p>Membrane tube networks were pulled from Golgi-mix GUVs containing 1% red fluorescent lipid BodTRCer and 1% biotinylated lipid Biot-CAP-PE by a truncated biotinylated version of kinesin1. Tube networks (red panel) were incubated with (A) 1 µM Arfaptin2-Alexa⁴⁸⁸, (B) 1 µM Arfaptin2-Alexa⁴⁸⁸ and 1 µM Arf1:GDP, or (C) 1 µM Arfaptin2-Alexa⁴⁸⁸ and 1 µM Arf1:GTPγS. Arrows ending with a triangle point to highly curved membrane regions (tubes) where Arfaptin2 is bound. Arrows ending with a circle point to weakly curved membrane regions (vesicles) where Arfaptin2 binding is not detected. Scale bar: 10 µm.</p