Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion

Jarungjit Rujiviphat,Gabriela Meglei,John L Rubinstein,G.Angus Mcquibban

doi:10.1074/jbc.m109.044933

Jarungjit Rujiviphat, Gabriela Meglei + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.044933

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Abstract

Mgm1, the yeast ortholog of mammalian OPA1, is a key component in mitochondrial membrane fusion and is required for maintaining mitochondrial dynamics and morphology. We showed recently that the purified short isoform of Mgm1 (s-Mgm1) possesses GTPase activity, self-assembles into low order oligomers, and interacts specifically with negatively charged phospholipids (Meglei, G., and McQuibban, G. A. (2009) Biochemistry 48, 1774-1784). Here, we demonstrate that s-Mgm1 binds to a mixture of phospholipids characteristic of the mitochondrial inner membrane. Binding to physiologically representative lipids results in approximately 50-fold stimulation of s-Mgm1 GTPase activity. s-Mgm1 point mutants that are defective in oligomerization and lipid binding do not exhibit such stimulation and do not function in vivo. Electron microscopy and lipid turbidity assays demonstrate that s-Mgm1 promotes liposome interaction. Furthermore, s-Mgm1 assembles onto liposomes as oligomeric rings with 3-fold symmetry. The projection map of negatively stained s-Mgm1 shows six monomers, consistent with two stacked trimers. Taken together, our data identify a lipid-binding domain in Mgm1, and the structural analysis suggests a model of how Mgm1 promotes the fusion of opposing mitochondrial inner membranes.

Highlights

S-Mgm1 is peripherally associated with the IM and found in the intermembrane space. short isoform of Mgm1 (s-Mgm1) results from the regulated cleavage by the mitochondrial rhomboid protease [7, 8]
Lipid-binding Domain within s-Mgm1—In our previous study [6], s-Mgm1 interactions with single phospholipids were assayed by lipid overlay Western blotting
We included Lys854 in our analyses, as this residue is known to be required for s-Mgm1

Summary

Introduction

S-Mgm1 is peripherally associated with the IM and found in the intermembrane space. s-Mgm1 results from the regulated cleavage by the mitochondrial rhomboid protease [7, 8]. We have shown previously that the dynaminrelated protein Mgm1 has GTPase activity, self-assembles into low order oligomers, and binds to negatively charged phospholipids [6]. Phospholipids Stimulate Mgm1 Activity In Vivo Complementation Assay and Fluorescence Microscopy— Plasmid shuffle complementation and imaging of mitochondrial morphology were performed as described previously [8].

Results

Conclusion