Golgi protein FAPP2 tubulates membranes

Abstract

The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network.

Fig. 1.

FAPP2-mediated tubulation of flat membrane sheets. Tubulation activity of FAPP2 on membrane sheets with different compositions was followed by DIC. Lipids membrane sheets consisted of the following: (A) POPC:PI (4)P:GlcCer (96:2:2 mol%); (B) POPC:PI (4)P (98:2 mol%); (C) POPC; and (D) POPC:GlcCer (98:2 mol%). (E) Fluorescence and dark field images of tubules generated on membrane sheet containing POPC: PI (4)P (98:2 mol%) by mCherry-FAPP2. Tubulation was initiated by injection of 5 μl FAPP2 (1 mg/ml) into the reaction chamber. Bars, 40 μm.

Fig. 2.

Membrane tubulation activity of FAPP2 is PI (4)P dependent. Screen shots were taken from Movies S1–S3. (A) PI (4)P binding–deficient FAPP2-R18L lacks tubulation activity, whereas addition of WT-FAPP2 rescues membrane tubulation (B). (C) FAPP2-W407A, lacking GlcCer binding, displays tubulation activity as WT-FAPP2. Fast and narrow tubules, indicated by arrows, are appearing at early time point as in Fig. 1A but grow back toward the lipid sheet. In all tubulation assays, a lipid mixture consisting of POPC:PI (4)P:GlcCer (96:2:2 mol%) was used. Tubulation was initiated by injection of 5 μl FAPP2 (1 mg/ml) into the reaction chamber.

Fig. 3.

SAXS analyses of dimeric 3myc-FAPP2-His₆ protein. (A) Experimental scattering curve (blue) and theoretical scattering curve of the modeled envelope structure (red). (B) Distance distribution function shows 3myc-FAPP2-His₆ to be an extended molecule ≈30 nm in length. (C) Low-resolution envelope of 3myc-FAPP2-Hi_s calculated by ab initio modeling (blue mesh surface). Rigid body modeling was used to calculate the possible locations of the PH- (blue and orange) proline-rich (light green) and GLTP (dark green and red) domain of FAPP2.