Abstract
Mitochondria are double-membrane organelles that continuously undergo fission and fusion. Outer mitochondrial membrane fusion is mediated by the membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), carrying a GTP hydrolyzing domain (GTPase) and two coiled-coil repeats. The detailed mechanism on how the GTP hydrolysis allows Mfns to approach adjacent membranes into proximity and promote their fusion is currently under debate. Using model membranes built up as giant unilamellar vesicles (GUVs), we show here that Mfn1 promotes membrane adhesion of apposing lipid vesicles. The adhesion forces were sustained by the GDP-bound state of Mfn1 after GTP hydrolysis. In contrast, the incubation with the GDP:AlF, which mimics the GTP transition state, did not induce membrane adhesion. Due to the flexible nature of lipid membranes, the adhesion strength depended on the surface concentration of Mfn1 through a cooperative binding mechanism. We discuss a possible scenario for the outer mitochondrial membrane fusion based on the modulated action of Mfn1.
Highlights
The fusion and fission of biomembranes are important events within the eukaryotic cell
Though the overproduction of Homo Sapiens Mitofusin 1 (hsMfn1) was not appreciated on SDS-PAGE (Figure 1A, left), Western blotting using the specific mitofusin 1 (Mfn1) antibody showed that hsMfn1 was present at 60 min and 90 min upon the induction with Isopropyl β-D-1-thiogalacto-pyranoside (IPTG), but is degraded at later time points (Figure 1A, right)
At 100 min the amount of GTP in the sample must have been depleted as no significant increase of the amount of Pi was observed with respect to 60 min of incubation. These results suggest that the heterologously produced hsMfn1 was proficient for GTP hydrolysing activity, but as we needed a high concentration (10 mM) of nucleotide to observe the GTP hydrolysis we must conclude that our hsMfn1 protein either bound or hydrolyzed the nucleotide with a low affinity
Summary
The fusion and fission of biomembranes are important events within the eukaryotic cell. Membrane remodelling needs to overcome various energy barriers, where the target membranes must first be brought into very close proximity and be destabilised to merge their lipid bilayers. DLPs are membrane active GTP hydrolysing proteins (GTPases) with high structural similarity, but low sequence identity that operate in different organelles within the eukaryotic cell where they trigger vesicle formation, membrane fusion, or organelle division [4,6,7]. ATL is a bifunctional protein that catalyses the docking (or adhesion) of the ER membranes through a GTP-dependent dimerization of its GTPase domain originating from adjacent membranes (trans), whereas the membrane fusion event is mediated by its carboxy-terminal (C-terminal) amphipathic helix that destabilises the lipid bilayer [8]
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