MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins

Abstract

Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the ‘Mitochondrial Contact Site and Cristae Organising System’ (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function.

Mitochondrial Apolipoprotein MIC26 is a metabolic rheostat regulating central cellular fuel pathways

“…Cells were grown at 37°C supplied with 5% CO 2 . MIC26 HepG2 KO cells were generated using the double nickase method as described before (Lubeck et al ., 2023). Cells cultured in standard growth media were divided equally into two cell culture flasks and grown in either 1 g/L glucose DMEM (normoglycemia) or 4.5 g/L glucose DMEM (hyperglycemia) (PAN-Biotech) supplemented with above-mentioned reagents.…”

“…However, the recombinant protein was only observed at the expected size of 22 kDa (Lamant et al ., 2006) and it was later shown that this 22 kDa form is located to mitochondria (Koob et al ., 2015; Ott et al , 2015). Moreover, using several cellular MIC26 deletion models and different antibodies, we showed recently that MIC26 is exclusively present as a 22 kDa mitochondrial protein and not as a 55 kDa protein (Lubeck et al , 2023). In light of these findings, the primary physiological function of MIC26 in diabetes is linked to its role in the mitochondrial IM and not to an earlier proposed secreted form of MIC26.…”

A X‐linked nonsense APOO/MIC26 variant causes a lethal mitochondrial disease with progeria‐like phenotypes

“…The 55 KDa band was present even in the KO of MIC26 and mass spectrometry of 55 KDa protein did not contain any MIC26-specific peptides, showing that the 55 KDa band was nonspecific. 39 MIC26 and MIC27 show antagonistic regulation at the protein level where a decrease in the levels of one of them leads to an increase in protein levels of another and vice versa. 38 Although they are not considered as core components of MICOS complex, the double KO of MIC26 and MIC27 shows drastic loss of CJs.…”