In this report, we characterize a novel vertebrate-specific mitochondrial outer membrane protein MIEF1, which regulates mitochondrial morphology. Overexpression of MIEF1 shifts the balance towards a fusion phenotype, characterized by extensively elongated mitochondrial tubules and perinuclear tubular clusters, whereas knockdown of MIEF1 leads to mitochondrial fragmentation. MIEF1 binds to and recruits Drp1 to the mitochondrial surface. Despite the fact that MIEF1 recruits Drp1 to mitochondria, it negatively affects the fission-promoting capacity of Drp1 by sequestering and inhibiting Drp1 activity, resulting in the mitochondrial fusion phenotype. To our knowledge, MIEF1 is the first Drp1 suppressor protein identified in vertebrates that efficiently impedes Drp1-mediated fission. Multiple lines of evidence support that MIEF1 has a key role in inhibition of Drp1-mediated mitochondrial fission. Firstly, MIEF1–Drp1 binding and the MIEF1-mediated recruitment of Drp1 to mitochondria do not require hFis1, Mff and Mfn2 (), as well as are independent of Drp1 GTPase activity () or phosphorylation status. MIEF1 can efficiently bind to wild-type Drp1 as well as the Drp1S637A
mutants (Supplementary Figure S4
). Moreover, MIEF1 overexpression does not affect the phosphorylation level of Drp1 (). Secondly, MIEF1 overexpression reduces the GTP-binding levels of both endogenous Drp1 and exogenous HA-Drp1 (). This suggests that MIEF1 may affect the GTPase activity of Drp1 via a reduction of its GTP-binding ability. Thirdly, the MIEF1 mutant MIEF1Δ1−48
, which is cytoplasmically localized but retains Drp1-binding ability and sequesters Drp1 in the cytoplasm, causes a mitochondrial fusion phenotype similar to that induced by Drp1K38A
or by knockdown of Drp1
mRNA by RNAi ( and ). Conversely, the MIEF1 mutant MIEF1Δ160−169
, which is membrane tethered but does not bind to Drp1, does not cause the mitochondrial fusion phenotype (; Supplementary Figure S3
). Collectively, the findings presented in this work shed light on the longstanding question of how Drp1 translocates from the cytoplasm to mitochondria and how Drp1-mediated fission is regulated in vertebrates (Hoppins et al, 2007
; Santel and Frank, 2008
; Liesa et al, 2009
; Westermann, 2010b
). Consistent with our findings, another group characterized the same cDNA of MIEF1(MiD51) and published their findings during the editorial processing of this work. They showed that MIEF1/MiD51 recruits Drp1 to mitochondria and that overexpression of MIEF1/MiD51 induces mitochondrial fusion, confirming a role for MIEF1 in inhibition of Drp1-mediated mitochondrial fission (Palmer et al, 2011
). The function of MIEF1 is distinct from the recently characterized vertebrate-specific Mff protein (Gandre-Babbe and van der Bliek, 2008
; Otera et al, 2010
). While Mff, like MIEF1, recruits Drp1 to mitochondria, overexpression of Mff leads to a fission phenotype, whereas Mff knockdown promotes fusion. We therefore suggest that MIEF1 and Mff have opposite effects on mitochondrial fission. MIEF1 has a key role in inhibiting Drp1-induced mitochondrial fission, while Mff has a role in promoting Drp1-induced mitochondrial fission. Therefore, Mff and MIEF1 positively and negatively regulate Drp1-mediated mitochondrial fission in vertebrates, respectively.
Mitochondrial fusion is thought to be initiated by Mfn-mediated mitochondrial tethering. Tethering of adjacent mitochondria is required for bringing membranes into close apposition for consequent membrane fusion events, where the GTPase activity of Mfn may function downstream of mitochondrial tethering to mediate full fusion (Koshiba et al, 2004
). However, it seems plausible that MIEF1, in addition to blocking Drp1 fission activity, also actively promotes fusion. Notably, overexpression of MIEF1 can induce a high proportion of cells to exhibit a compact cluster of mitochondria, while inhibition of mitochondrial fission via MIEF1Δ1−48
, or via depletion of either Drp1 or Mff does not induce a highly compact cluster phenotype (see ). This suggests that MIEF1 has the capacity to bring the mitochondrial membranes into close apposition and ultimately facilitates the process of membrane fusion as revealed by EM in . Moreover, the data from an in vivo
cell fusion assay reveal that MIEF1 overexpression promotes mitochondrial fusion. Conversely, depletion of endogenous MIEF1 results in partially fragmented mitochondria. Two lines of evidence indicate that MIEF1 promotes fusion in a manner independent of the fusion-promoting GTPase Mfn2. Firstly, the mitochondrial fusion phenotypes caused by overexpression of MIEF1 and Mfn2 are not identical, and secondly, MIEF1 is capable of inducing mitochondrial fusion also under conditions when Mfn2 expression is knocked down by RNAi. The accumulation of MIEF1 into discrete puncta at the connection sites of adjacent mitochondrial units, along with the observation that MIEF1 undergoes oligomerization by self-association, indicates that interactions between MIEF1 proteins on opposing mitochondrial membranes may contribute to the fusion process.
hFis1 is a fission-promoting protein, which was proposed to serve as a receptor for recruitment of Drp1 to mitochondria (James et al, 2003
; Yoon et al, 2003
). In yeast, Fis1p (the hFis1 homologue) interacts with and recruits Dnm1p (the Drp1 homologue) to mitochondria through the yeast-specific adaptor proteins Mdv1p or Caf4p (Mozdy et al, 2000
; Tieu and Nunnari, 2000
; Griffin et al, 2005
). The fact that elevated expression of hFis1, in contrast to MIEF1, does not relocalize Drp1 to mitochondria argues against a direct role for hFis1 in the recruitment process in vertebrates. Furthermore, hFis1 localizes evenly throughout the mitochondrial surface (Suzuki et al, 2003
), whereas MIEF1 and Drp1 colocalize as discrete puncta extensively. MIEF1 shows a robust interaction with hFis1, in contrast to a much weaker interaction observed between hFis1 and Drp1. The MIEF1–hFis1 interaction is likely to be of functional significance, as elevated hFis1 partially reverts the MIEF1-induced fusion phenotype. While it may be tempting to speculate that MIEF1, because it can interact with Drp1 and hFis1 through non-overlapping domains, would serve as a bridging protein between Drp1 and hFis1, our data indicate that the MIEF1–Drp1 and MIEF1–hFis1 interactions are independent. Firstly, the binding between hFis1 and Drp1 is not affected by elevated levels of MIEF1 or MIEF1 mutants. Secondly, depletion of hFis1 or Drp1 by RNAi does not affect MIEF1 interaction with Drp1 and hFis1, respectively. Thirdly, the native gel electrophoresis (NGE) shows that the complexes containing MIEF1 and Drp1 differ in size from those containing MIEF1 and hFis1. Assuming that MIEF1 independently interacts with hFis1 and Drp1, it may be argued that the relative levels of hFis1 and MIEF1 regulate Drp1-mediated mitochondrial fission, such that high levels of MIEF1 promote a MIEF1–Drp1 interaction, which inhibits Drp1-mediated fission, leading to mitochondrial fusion, whereas high levels of hFis1 reverse the MIEF1-induced fusion phenotype, thus leading to mitochondrial fission.
The characterization of MIEF1 also highlights that mitochondrial morphology is differently regulated in vertebrates and yeast. We suggest that the molecular machinery controlling mitochondrial dynamics has evolved such that two of the central components, the Dmn1p/Drp1 and Fis1p/hFis1 proteins, are highly conserved in both yeast and vertebrates, while the interacting proteins, that is MIEF1 and Mff versus Mdv1p and Caf4p, are quite evolutionarily and functionally diverged.
In conclusion, the characterization of MIEF1 sheds new light on how the molecular machinery controlling mitochondrial morphology in mammals is organized and also highlights important differences between yeast and mammals in the composition of this machinery. Given the importance of mitochondrial dynamics for many cellular processes, the discovery of a novel key regulator protein in the process may provide further options to experimentally steer the process, which may be of importance in understanding the role of mitochondrial dynamics in cancer, diabetes, cardiovascular and neurodegenerative diseases.