Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

Abstract

The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

Fig. 1

Production of GTPase active AtDRP1A. (A) SDS–PAGE gel analysis of E. coli expressed, amylose resin purified, His₈-MBP-AtDRP1A and AtDRP1A prior to (lane 1) and after (lane 2) cleavage of His₈-MBP with TEV protease. Proteins were detected by Coomassie staining. M = Molecular Mass Standards. (B) The GTPase activity of purified AtDRP1A, His₈-MBP-AtDRP1A and a mixture of His₈-MBP and His₆-TEV (negative control) was assayed with a colorimetric assay to measure released phosphate. (C) The activity of TEV-cleaved AtDRP1A at various concentrations of GTP was used to calculate the kCat and kM for AtDRP1A.

Fig. 2

AtDRP1A forms large polymers in vitro. (A) Purified His₈-MBP -AtDRP1A and TEV-cleaved AtDRP1A (200 nM) was centrifuged at 150,000g in a pH 7.5 HEPES buffer containing 150 mM NaCl and 2 mM MgCl₂. The load and supernatant (S150) were analyzed by immunoblotting with antibodies against AtDRP1A and MBP. The immunoblot was overexposed to demonstrate absence of AtDRP1A in the S150. (B) AtDRP1A (50 nM) was separated on a 5–50% (w/v) sucrose gradient by centrifugation at 150,000g for 18 h and gradient fractions were analyzed by immunoblotting with antibodies against AtDRP1A. Black and gray bars represent the peak and range, respectively, for three molecular weight markers (BSA 4.4S, Catalase 11.4S and AtCDC48 17S) run in parallel on a separate gradient and detected by SDS–PAGE gel analysis and Coomassie staining. Equivalent fractions were determined by measurement of refractive index. (C) AtDRP1A (1 µM) visualized by electron microscopy after negative staining with Nano-W^®. Right panels are higher magnification views. Arrowheads indicate smaller AtDRP1A polymers; arrows indicate larger AtDRP1A polymers. Scale bars = 100 nm.

Fig. 3

AtDRP1A binds and clusters protein-free liposomes. (A–B) Liposome flotation assay. (A) Arabidopsis Plasma Membrane Mimetic liposomes (“PMM”: 40% β-sisterol, 25% Soy PC, 20% DOPE, 10%DOPS and 5% DOPG) or (B) neutrally charged liposomes (“Neutral”: 40% β-sisterol, 40% Soy PC, 20% DOPE) spiked with trace H³-DOPC were generated by extrusion through a 50 nm membrane. These liposomes (44 mM) were incubated with purified AtDRP1A (250 nM) and separated from the load by flotation on a 40–30–0% (w/v) Accudenz step-gradient. The two top (1, 2) and the two bottom (6, 7) fractions of the gradient were analyzed by scintillation counting and immunoblotting with antibodies against AtDRP1A and MBP. (C) PMM liposomes were stained with OsO₄ followed by Nano-W^® and visualized by electron microscopy. (D) PMM liposomes were incubated with AtDRP1A then stained and visualized as in (C). Arrows indicate clusters of liposomes induced by binding of AtDRP1A. Scale bars in (C–D) = 100 nm.