Plant mitochondrial dynamics and the role of membrane lipids

Abstract

Mitochondria are highly dynamic organelles that are continuously shaped by the antagonistic fission and fusion processes. The major machineries of mitochondrial fission and fusion, as well as mechanisms that regulate the function of key players in these processes have been analyzed in different experimental systems. In plants however, the mitochondrial fusion machinery is still largely unknown, and the regulatory mechanisms of the fission machinery are just beginning to be elucidated. This review focuses on the molecular mechanisms underlying plant mitochondrial dynamics and regulation of some of the key factors, especially the roles of membrane lipids such as cardiolipin.

Eukaryotic cells contain membrane-enclosed subcellular organelles. Among them, the double-membrane mitochondria are descendants of ancient endosymbiotic α-proteobacteria and serve as the power house of the cell because of their role in supplying the biological energy ATP.^1,2 In plants, mitochondria participate in many vital physiological processes, such as energy metabolism, programmed cell death, intracellular calcium homeostasis, primary carbon metabolism, amino acid metabolism and the biosynthesis of lipids and vitamins.³ Two other essential organelles, i.e. peroxisomes and chloroplasts, function coordinately with mitochondria in plant energy capture, conversion, storage and/or metabolism.^3-5

Mitochondria are highly dynamic, as they can change in number, morphology, distribution, and protein and lipid compositions in response to developmental and pathological needs or environmental cues. Mitochondria are continuously shaped by the antagonistic fission and fusion events. The major machineries of mitochondrial fission and fusion, as well as mechanisms that regulate the function of key players in these processes, have been identified from studies in different experimental systems, including mammals, yeasts and plants.^6,7 This review focuses on studies from plants on the cellular and molecular mechanisms that govern mitochondrial dynamics. Where applicable, differences between plants and other eukaryotic systems and evidence obtained from other systems but still missing from plants will also be discussed.

The Plant Mitochondrial Fission Machinery

In Arabidopsis, the mitochondrial fission machinery is composed of conserved proteins such as Dynamin-Related Protein 3 (DRP3) and Fission1 (FIS1), as well as plant-specific factors such as Elongated Mitochondria1 (ELM1) and Peroxisomal and Mitochondrial Division factor1 (PMD1) (Fig. 1). Interestingly, with the exception of ELM1, these proteins are shared by the mitochondrion and its metabolically linked sister organelle, the peroxisome.

Figure 1.

The protein machinery involved in mitochondrial fission in Arabidopsis. Dynamin-Related Protein 3A (DRP3A) and DRP3B are large GTPases that assemble into ring structures at the organelle fission sites. FISSION1A (FIS1A) and FIS1B are C-terminal anchored membrane proteins possibly responsible for DRP3 recruitment to the organelles. Peroxisome and Mitochondrial Division factor 1 (PMD1) functions independently of the FIS1-DRP3 complex in promoting mitochondrial division. Elongated Mitochondria 1 (ELM1) is a mitochondrial specific fission factor involved in DRP3 recruitment to mitochondria. Cardiolipin supports DRP3 function in mitochondrial fission through stabilizing DRP3 protein complexes. Only the mitochondrial outer membrane is depicted. For clarity, the membrane associated proteins and cardiolipin are only shown at one outer membrane. Arrows indicate interactions.

The function of the mitochondrial fission DRP is conserved in diverse species. Mitochondrial fission DRPs include DRP3A and DRP3B in Arabidopsis, hsDRP1 in human and scDNM1 in Saccharomyces cerevisiae.^8-13 Arabidopsis loss-of-function mutants of DRP3A and DRP3B contain markedly elongated mitochondria and peroxisomes.^8-11 The crystal structure of hsDRP1 reveals a GTPase domain, a bundle signaling element and a stalk.¹⁴ These cytosolic large GTPases dynamically localize at the organelle fission sites, where they form a ring-like structure via higher order assembly (Fig. 1) and promote membrane fission through assembly- and GTP hydrolysis-driven constrictions.^15-19 FIS1 proteins are also conserved across kingdoms, participating in the recruitment of DRPs from the cytosol to organelle membranes (Fig. 1).^11,20-26 Interestingly, Arabidopsis FIS1A also localizes to chloroplasts,²⁷ indicating its possible role in chloroplast dynamics.

The plant-specific fission factor Elongated Mitochondria1 (ELM1) is localized at the surface of mitochondria and physically interacts with DRP3, responsible for the translocation of DRP3 to mitochondria in a FIS1-independent manner (Fig. 1).²⁸ Another plant-specific fission factor is PMD1, which acts independently from the FIS1-DRP3 complex with an unknown mechanism (Fig. 1).²⁹

In S. cerevisiae, 2 WD40-repeat domain-containing proteins MDV1 and CAF4 function as adaptors between scFIS1 and scDNM1, physically interacting with scFIS1 and scDNM1 as well as themselves.^26,30-32 In metazoans, the coiled-coil domain-containing membrane protein Mitochondrial fission factor (Mff) interacts with DRP1 and serves as DRP1's receptor.^33,34 Moreover, metazoan MiD49 and MiD51 interact with DRP1 and FIS1, mediating the recruitment of DRP1 to mitochondria in the absence of Mff and FIS1.^35-39 Therefore, different mechanisms are involved in the localization of DRP1 and DNM1 to mitochondria. MDV1, CAF4, Mff, MiD49 and MiD51 do not have apparent orthologs in plants.

Mitochondrial Fusion Proteins: Still Largely Unidentified in Plants

Animal and yeast mitochondria often appear highly tubular and interconnected, which reflects the dominance of mitochondrial fusion in these organisms.⁴⁰ The mitochondrial fusion machineries are well characterized in these systems with the core components being members of the DRP family as well.⁷ The mammalian mitochondrial fusion DRPs are mitofusin 1 and 2 (MFN1/2) on the outer membrane^41,42 and optic atrophy1 (OPA1) on the inner membrane.^43-45 Their yeast counterparts are FZO1⁴⁶ and MGM1^47-50 respectively.

In contrast, plant mitochondria are highly fragmented under normal conditions, suggesting that plant mitochondrial dynamics is fission dominant. However, mitochondrial fusion does occur in plants. In a fluorescence mixing experiment, where the mitochondrion-targeted photoconvertible fluorescent protein Kaede was used in onion bulb epidermal cells and first converted from green to red in a sub-population of the mitochondria, yellow mitochondria resulted from the mixing of green and red Kaede fluorescent proteins were observed, indicating mitochondrial fusion events.⁵¹ Extensive mitochondrial fusion prior to cell division was reported in cultured protoplasts,⁵² and reticular mitochondria were observed during G1 to S phase of the cell cycle in Arabidopsis shoot apical meristem,⁵³ suggesting that mitochondrial fusion may be more prominent at certain stages of the cell cycle in plants. The Arabidopsis protein FRIENDLY was suggested to play a role in mediating inter-mitochondrial association, an important step prior to fusion.⁵⁴ However, the core components of the plant mitochondrial fusion apparatus remain to be discovered.

Post-Translational Regulation of Mitochondrial Dynamics

With respect to the regulatory mechanisms of mitochondrial fission and fusion, post-translational modifications (PTMs) of the mammalian mitochondrial DRP1 are best characterized. These PTMs include phosphorylation, SUMOylaiton, ubiquitination, and S-Nitrosylation, which result in the modulation of DRP's dimerization/oligomerization, GTPase activity, localization to and departure from mitochondria, or protein stability.^7,55

The knowledge about post-translational regulation of Arabidopsis DRP3 is still limited. Multiple genome-wide proteomic experiments identified phosphorylation sites at various positions on DRP3,^56-61 yet the consequences of these phosphorylation events have not been determined yet. DRP3A and DRP3B were also reported to undergo mitotic phosphorylation and ubiquitination at unidentified sites, affecting mitochondrial fission during mitosis.⁶² Recently, the Arabidopsis mitochondrial outer membrane-anchored ubiquitin-specific protease UBP27 was discovered to be involved in mitochondrial dynamics.⁶³ Whether UBP27 directly targets the mitochondrial fission/fusion proteins needs to be further analyzed.

The Role of Phospholipids in Mitochondrial Dynamics

The roles of diverse membrane lipids in mitochondrial dynamics are also beginning to be elucidated. Lipids can play structural roles in membrane remodeling process, such as the formation of membrane curvature (Fig. 2A).^64-67 They may also interact with proteins directly, recruiting proteins to the membranes or affecting protein activities.^68-70

Figure 2.

Lipid shapes and the structure of cardiolipin (CL). (A) Lipid molecules with different conicities. Cone shaped and inverted cone shaped lipids are considered non-bilayer forming lipids, because they can form single layer membrane structures in vitro. When non-bilayer forming lipids are incorporated into bilayer membranes, they tend to induce membrane curvatures. (B) Molecule structure of CL, which is dimeric and contains 4 acyl groups and potentially 2 negative charges.

Cardiolipin (CL) is considered as a signature lipid of mitochondria. It is an anionic phospholipid with a dimeric structure and contains a triple glycerol backbone and 4 acyl groups. CL has a conical shape, with the head group smaller than the acyl chains. Hence, CL belongs to the so-called non-bilayer forming lipids, which promote membrane curvature and possibly play a structural role in membrane fission and fusion (Fig. 2A and B).⁷¹

In Arabidopsis, CL was shown to be important for mitochondrial fission. Disruption of Cardiolipin Synthase (CLS) results in strong mitochondrial elongation and enlargement.^72,73 CL's function in mitochondrial fission was shown to be, at least in part, through stabilizing the protein complex of DRP3 (Fig. 1).⁷² In addition, CL is involved in maintaining mitochondrial ultra-structure and in plant responses to stresses that induce programmed cell death.^72,73 In support of the plant work was the finding that in mammals, CL can interact with the organelle fission factor DRP1 in vitro, inducing DRP1 oligomerization, assembly-stimulated GTP hydrolysis and DRP1's ability to promote membrane tubulation.^74,75 CL also enhances mitochondrial fission by recruiting α-Synuclein, a Parkinson disease-related protein that promotes mitochondrial fission, to the mitochondrial membrane in mammalian cells.⁷⁶

In contrast to plant CL that serves a dominant role in mitochondrial fission, CL appears to be primarily a pro-fusion factor at least in yeast cells. CL deficiency in yeast cells that lack CL's positive regulator UPS1 causes mitochondrial fragmentation.⁷⁷ CL is functionally related to the mitochondrial fusion DRPs MGM1 and OPA1. It interacts with MGM1 to stimulate its GTPase activity and dimerization. The enzymatic processing of MGM1 to generate the short isoform of MGM1 (s-MGM1), which is required for full MGM1 activity in mitochondrial fusion, is dependent on CL.^77-79 The mammalian OPA1 also interacts with liposomes that contain acidic phospholipids, such as CL, phosphatidic acid (PA) and phosphatidylserine (PS), resulting in the stimulation of OPA1s GTPase activity.⁸⁰ CL is much more abundant than PA and PS in mitochondrial membranes,⁸¹ suggesting that CL may play a role in supporting OPA1 activity in fusion as well.

Although not yet reported in plants, several other lipid species in the mitochondrial membrane have been shown to play pivotal roles in mitochondrial dynamics in mammals and yeasts. For example, phosphatidylethanolamine (PE) is another major non-bilayer forming lipid in the mitochondrial membrane. It regulates mitochondrial fusion possibly through modulating membrane biophysical properties and MGM1 processing, and is partially redundant to CL's functions.^81,82 Phosphatidic acid (PA) is a negatively charged and cone-shaped phospholipid, similar to CL, that serves as an important structural lipid in promoting the formation of negative membrane curvature, thus playing a positive role in mitochondrial fusion.^83,84 PA can be generated through the cleavage of CL by Mitochondrial surface Phospholipase D (MitoPLD),^70,85,86 and may exert its function through the fusion DRPs MFN1 and MFN2.⁴² Lysophosphatidic acid (LPA)⁸⁷ and diacylglycerol (DAG)^69,88 are also involved in mitochondrial dynamics, yet their exact mode of action needs further elucidation.

Conclusions and Future Perspectives

Mitochondrial abundance and morphology are determined by the equilibrium of fission and fusion. In Arabidopsis, the mitochondrial fission machinery is well characterized and the major components largely conserved in other kingdoms. However, the core plant mitochondrial fusion machinery is still unidentified. Studies in mammals and yeasts have uncovered intricate mechanisms that regulate the components of the protein machinery involved in mitochondrial dynamics, including post-translational modifications and the action of membrane phospholipids. However, such mechanisms are just beginning to be explored in plants.

The interaction between lipids and proteins may provide a basis for membrane associated protein functions. Lipids may also play a structural role during membrane re-structuring. CL deficiency causes defects in mitochondrial fission in Arabidopsis. However, CL may also be involved in plant mitochondrial fusion, but this seems to be hard to analyze without the identification of the fusion proteins. A quantitative analysis of the mitochondrial fusion rate in CL deficient mutant cells may determine whether there is a delay in mitochondrial fusion. Other phospholipids, such as PE, PS and PA, may also play a role in regulating plant mitochondrial fission and fusion. In addition, the biosynthetic and especially the remodeling pathways of phospholipids can impact mitochondrial dynamics in other systems.^89,90 Hence, investigating mitochondrial phospholipid remodeling pathways is an important future direction for the study of mitochondrial dynamics in plants.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

Work in the Hu lab on organelle dynamics was supported by the National Science Foundation (MCB 1330441).