Characterization of mitochondrial membrane proteins as novel components in mitochondrial dynamics

Mitochondria are dynamic organelles that frequently change their shape by shifting the balance of fusion and fission in response to cellular metabolic needs. This process is termed mitochondrial dynamics. Dysregulation of mitochondrial dynamics has been described in many human diseases. Mitochondrial dynamics has been widely studied in yeast, while in mammals the proteins involved in mitochondrial fusion and fission are divergent to some extent. To better understand the mechanisms in mammals, it is imperative to discover the key proteins underlying mitochondrial dynamics.

In this thesis, we characterized three novel mitochondrial membrane proteins, MIEF1, MIEF2 and MTGM (Romo1), which play important roles in mitochondrial dynamics. In paper i, MIEF1 is characterized as an integral mitochondrial outer membrane protein. Overexpression of MIEF1 induced extensive mitochondrial fusion, whereas depletion of MIEF1 caused mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria independent of hFis1, Mff and Mfn2 but this results in mitochondrial fusion. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reversed the MIEF1-induced fusion phenotype. MIEF1-induced mitochondrial fusion occurs in a manner distinct from Mfn2.

In paper ii, we compared MIEF1 and MIEF2, which are two paralogs in human. MIEF1 and MIEF2 possess many functions in common in mitochondrial dynamics. They anchor in the mitochondrial outer membrane, recruit Drp1 to mitochondria, and impede mitochondrial fission. They interact with Drp1 and hFis1. To some extent, MIEF1 and MIEF2 may play functionally distinct roles in mitochondrial dynamics. They are differentially expressed in human tissues. Ectopic expression of MIEF2 displayed a stronger mitochondrial fusion effect than MIEF1. hFis1 and Mff partially reverted MIEF2-induced fusion in contrast to the extensive rescue of fusion induced by MIEF1. MIEF2 forms higher order oligomers, while MIEF1 mainly presents as dimers. By studying engineered deletion mutants, it was shown that MIEF1 requires the amino acid residues 109-154 and MIEF2 the amino acids 1-49 for their dimerization/oligomerization. In MIEF1, oligomerization is not required for mitochondrial localization and interaction with Drp1.

In paper iii, gene expression profiling indicated that MTGM might be related to human brain tumors. We identified the highly conserved human MTGM gene that encodes an integral mitochondrial inner membrane protein and confirmed the upregulation of MTGM in human brain tumors. Overexpression of MTGM resulted in Drp1-dependent mitochondrial fragmentation and release of mitochondrial Smac/Diablo to the cytosol, but had no effect on apoptosis. Cell proliferation was inhibited by stalling of cells in S phase. Downregulation of MTGM induced mitochondrial elongation, an increase of cell proliferation and inhibition of cell death induced by apoptotic stimuli.

In paper iv, we discovered that the subcellular localization of NCX3 varies with the cell cycle phases. One phenotype is distribution of NCX3 in the plasma membrane (NCX3-PM), the other in the cytoplasm/ER (NCX3-ER). Modification of NCX3 by N-linked glycosylation of a single asparagine residue, N45, is normally required for targeting of the protein to the plasma membrane. Importantly, this modification also affects the cell cycle. In sum, this thesis has unveiled novel proteins playing pivotal roles in the regulation of mitochondrial dynamics.

List of scientific papers

I. Zhao, J., T. Liu, S. Jin, X. Wang, M. Qu, P. Uhlen, N. Tomilin, O. Shupliakov, U. Lendahl and M. Nister (2011). Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission. EMBO J 30(14): 2762-2778.
https://doi.org/10.1038/emboj.2011.198

II. Liu, T., R. Yu, S. B. Jin, L. Han, U. Lendahl, J. Zhao and M. Nister (2013). The mitochondrial elongation factors MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics. Exp Cell Res. 319(18): 2893- 2904.
https://doi.org/10.1016/j.yexcr.2013.07.010

III. Zhao, J., T. Liu, S. B. Jin, N. Tomilin, J. Castro, O. Shupliakov, U. Lendahl and M. Nister (2009). The novel conserved mitochondrial inner-membrane protein MTGM regulates mitochondrial morphology and cell proliferation. J Cell Sci. 122(Pt 13): 2252-2262.
https://doi.org/10.1242/jcs.038513

IV. Liu, T., J. Zhao, C. Ibarra, P. Uhlén, M. Nister. Glycosylation determines sodium-calcium exchanger 3 subcellular distribution during cell cycle; effects on ER Ca2+ handling. [Manuscript]