Control of mitochondrial dynamics by a fusogenic lipid

Abstract

Mitochondrial fusion enables cooperation between the mitochondrial population and is critical for mitochondrial function. Phosphatidic acid on the mitochondrial surface plays a key role in mitochondrial fusion. A recent study by Su et al. shows that the nucleoside diphosphate kinase NME3 recognizes PA and mediates its effects on mitochondrial dynamics.

Research in the last two decades has greatly advanced our understanding of how the dynamic properties of mitochondria regulate cellular physiology and pathology. In particular, mitochondrial fusion plays an essential role in maintaining mitochondrial physiology, exerting beneficial effects on the overall function of the mitochondrial population, tolerance of mitochondrial DNA damage, mitochondrial membrane potential, and response to cellular stress [1]. Key questions remain, however, regarding how mitochondrial fusion is regulated and how specific sites of fusion are selected and promoted. In 2006, phosphatidic acid was identified as a fusogenic lipid for mitochondrial fusion [2]. MitoPLD [mitochondrial phospholipase D, also called phospholipase D6 (PLD6)], catalyzes the conversion of cardiolipin to phosphatidic acid (PA) on the mitochondrial surface. This enzyme works synergistically with the mitochondrial GTP hydrolyzing enzymes Mitofusins 1 and 2 (Mfn1 and Mfn2) to promote mitochondrial outer membrane fusion. Intriguingly, overexpression of MitoPLD was found to promote aggregation of mitochondria, implying that PA can induce mitochondrial outer membranes to contact each other. Although the study revealed the importance of lipid composition in dynamically regulating fusion events, the mechanism of PA action and its precise relationship to mitofusins remained unclear. In particular, it was unclear how local deposition of PA by MitoPLD could promote adherence of mitochondrial membranes. In principle, PA could function as a signaling molecule or act to change the local physical properties of the membrane.

A recent study published in the Journal of Cell Biology by Su et al. reveals that the outer membrane protein NME3 is responsible for PA-induced mitochondrial aggregation (Figure 1) [3]. NME3 is a type of nucleoside diphosphate (NDP) kinase that was previously shown by the same group to be localized to mitochondria and physically interact with mitofusins to facilitate mitochondria fusion [4]. Two infants with a fatal neurodegenerative disorder characterized by hypotonia and defects in the cerebrum and cerebellum were found to have homozygous mutations in NME3 associated with reduced mitochondrial fusion and increased oxidative stress [4]. Patient cells were unable to tolerate glucose starvation, a stress condition that increases reliance on mitochondrial function. NDP kinases regulate the concentrations of nucleoside triphosphates, such as GTP and ATP, by exchanging the terminal phosphate between nucleoside diphosphates and nucleoside triphosphates. The NDP kinase NME4 has been implicated in promoting the activity of OPA1 by enhancing the local GTP concentration [5]. In contrast, the role of NME3 in mitochondrial fusion does not require its kinase enzymatic activity, indicating two distinct functions for NME3 [4].

Figure 1:

Multiple parameters affecting the sites of mitochondrial fusion. The Su et al study shows that hexameric NME3 is responsible for bringing together mitochondrial surfaces that have been decorated with PA by the action of MitoPLD. Mitochondria and endoplasmic reticulum show close contacts that are mediated by several protein complexes. These inter-organellar contact sites are favored sites of fusion due to the preferential localization of mitofusins, which use GTP hydrolysis to power membrane merger. Created in BioRender.

Following up on the Choi et al. study of 2006 [2], the Su et al. study shows that mitochondrial aggregation caused by MitoPLD overexpression is mediated by NME3. When NME3 is depleted, MitoPLD is no longer able to aggregate mitochondria. What is the basis for the ability of NME3 to aggregate PA-containing mitochondrial membranes? The N-terminal region of NME3 is hydrophobic and is required for the ability of NME3 to localize to mitochondria [3]. This conclusion was further supported by a liposome floatation assay, which showed that NME3 selectively associates with PA-containing liposomes, but not phosphatidylcholine- or cardiolipin-containing liposomes. Lowering the hydrophobicity of the N-terminal region reduces the membrane binding property of NME3. PA is known to favor formation of negatively curved membranes. In vesicle binding assays, NME3 showed binding to highly curved vesicles, even in the absence of PA. These results suggest that the N-terminal region of NME3 may preferentially associate with sites on the mitochondrial surface showing high curvature and containing PA. The ability of NME3 to aggregate mitochondria did not require its kinase activity, but did require its ability to form hexamers. Mutations disrupting hexamerization impeded mitochondrial aggregation, as well as aggregation of PA-containing liposomes.

To further clarify the role of NME3 in the timeline of fusion, the authors tracked NME3 localization and found that NME3 is enriched, in a MitoPLD-regulated manner, at the sites of mitochondrial contact before the fusion event happens. Interestingly, mitochondria-mitochondria contact sites showing NME3 enrichment have a higher probability of future fusion. These results suggest that NME3, via its hydrophobic N-terminus, dynamically associates with membranes containing PA deposited by MitoPLD. Because of its hexamerization property, NME3 accumulation at these sites would facilitate formation of mitochondria-mitochondria contacts, and such tethered structures have an enhanced tendency to progress to fusion. Although the model is attractive, it remains unclear why MitoPLD-induced mitochondrial aggregation in cells does not occur when both mitofusins are removed [2]. Nevertheless, MitoPLD, PA, and NME3 seem to dynamically regulate the sites where mitochondrial fusion occur. This type of regulation may also be involved in cell stress conditions that regulate mitochondrial dynamics, such as during the stress-induced hyperfusion that elongates mitochondria upon starvation [6]. Su et al. [3] found that nutrient deprivation increased the intensity of NME-GFP spots on mitochondrial contact sites, and the fusion probability of such sites was increased.

By uncovering the role of NME in recognizing PA and bringing mitochondrial membranes together, Su et al. clarify how the manipulation of membrane lipid composition can dynamically regulate mitochondrial membrane fusion. Key issues concerning the role of PA and NME3 in mitochondrial fusion remain to be resolved. It seems likely that the association of NME3 with mitochondria has to rely on more than its affinity for PA, given that PA is known to present in other intracellular organelles. Some other mitochondrial features, like mitochondria-targeting chaperones or OMM proteins, likely work with PA to bring NME3 to mitochondrial membranes. Consistent with this idea, the depletion of MitoPLD has little effect on endogenous NME3 localization. What happens after NME3 brings PA-containing mitochondrial membranes together? Experiments with overexpressed MitoPLD indicate that mitochondrial aggregation mediated by PA is insufficient for fusion. Mitofusins appear to be concentrated at mitochondria-endoplasmic reticulum contact sites (MERCS) to form preferred sites of membrane fusion [7]. NME3 has been shown to physically interact with both Mfn1 and Mfn2 [4]. The results of Su et al. [3] on NME3 provide motivation to focus on unraveling how the molecular relationships of NME3, PA, mitofusins and MERCS determine the specific sites for mitochondrial fusion.