Abstract
Every lipid membrane fission event involves the association of two apposing bilayers, mediated by proteins that can promote membrane curvature, fusion and fission. We tested the hypothesis that Fis1, a tail-anchored protein involved in mitochondrial and peroxisomal fission, promotes changes in membrane structure. We found that the cytosolic domain of Fis1 alone binds lipid vesicles, which is enhanced upon protonation and increasing concentrations of anionic phospholipids. Fluorescence and circular dichroism data indicate that the cytosolic domain undergoes a membrane-induced conformational change that buries two tryptophan side chains upon membrane binding. Light scattering and electron microscopy data show that membrane binding promotes lipid vesicle clustering. Remarkably, this vesicle clustering is reversible and vesicles largely retain their original shape and size. This raises the possibility that the Fis1 cytosolic domain might act in membrane fission by promoting a reversible membrane association, a necessary step in membrane fission.
Highlights
Peroxisomal and mitochondrial fission involve the cooperation of integral, peripheral, and soluble proteins to control organelle size, shape, and distribution [1]
We identify that Fis1 undergoes a membrane-induced conformational change that clusters lipid vesicles
The cytosolic domain of Fis1 (Fis1DTM) was shown to induce permeabilization of the small molecule ANTS from DOPC:DOPG (6:4) lipid vesicles at pH 5.0, but not at pH 7.0 [26]. To better understand this permeabilization, we first tested whether Fis1DTM could bind to lipid vesicles that mimic the mitochondrial outer membrane (Mitomix) [37]
Summary
Peroxisomal and mitochondrial fission involve the cooperation of integral, peripheral, and soluble proteins to control organelle size, shape, and distribution [1] The fission of these organelles appears to be executed by the same core machinery: a tailanchored integral membrane protein, Fis1 [2,3,4,5,6], and a cytosolic dynamin-like mechanoenzyme, Dnm (yeast) or Drp1/Dlp (mammals) [7,8,9]. Fis was initially discovered in yeast to be essential for the maintenance of mitochondrial homeostasis and is conserved in eukaryotes [2,3,4,5,16] This small, 17 kDa protein is uniformly localized on the cytosolic side of the mitochondrial outer membrane by a single-pass, C-terminal transmembrane domain, with a similar orientation on the peroxisomal surface [2,6]. Release of vesicle contents did not require the transmembrane domain and can be induced by the cytosolic domain of Fis alone, which raises the possibility that the cytosolic domain itself plays a role in altering membrane structure, independently of its accepted role in protein recruitment
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