These observations show that the 55 kDa protein does not originate from MIC26. == The mitochondrial MIC26 is consistently detected in murine tissues, cells and human cells in contrast to the putative 55 kDa glycosylated version == We have performedMIC26downregulation with three different siRNAs in HEK293 cells, probed with four different anti-MIC26 antibodies using control and HepG2MIC26KO cells and further testedMIC26KOs in three other human cell lines namely HEK293, HeLa and HAP1 cells (Fig 2), and exogenously expressed MIC26-GFP and MIC26-myc in HEK293 cells (Fig 3andS4 Fig) which consistently validated that the 55 kDa protein of MIC26 does not SU 5416 (Semaxinib) exist. 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. == Introduction == Mitochondria are essential double-membrane-enclosed organelles required for a myriad key cellular functions including ATP conversion, iron-sulfur cluster biogenesis, programmed cell death, calcium buffering, vital metabolic processes and inflammatory responses. The mitochondrial inner membrane (IM) is spatially heterogenous and composed of the inner boundary membrane (IBM), parallel to the outer membrane (OM), and the cristae membrane (CM) extending as invaginations towards the mitochondrial matrix. CMs house the electron transport chain (ETC) complexes [1,2] and are responsible for ATP generation. Cristae exist in a variety of shapes SU 5416 (Semaxinib) and sizes depending on the cell, tissue type and bioenergetic status of the cell [3,4]. Aberrant cristae are found in a wide variety of pathologies like diabetic cardiomyopathy, neurodegeneration and mitochondrial myopathy [57]. The IBM and CM are separated by distinct pore-like structures which are around 25 nm in diameter and termed crista junctions (CJs) [8,9]. CJs were proposed as a diffusion barrier for various metabolites, membrane and soluble proteins [1013]. In congruence, it was shown that there was differential localisation of various proteins in the IM: The IBM predominantly contains proteins involved in mitochondrial fusion and protein import while the CMs are enriched with proteins belonging to mitochondrial protein synthesis, iron-sulfur cluster biogenesis and ETC complexes [1,2]. In fact, the CJs give rise to heterogenous membrane potential of the individual cristae [14]. Therefore, CJs are an important structural and functional constituent of the IM. The MICOS (Mitochondrial contact site and cristae organising system) complex is a multi-protein complex comprising of sevenbona fideproteins which resides at the CJs. The MICOS complex was discovered inSaccharomyces cerevisiaeby independent groups [1517]. The proteins of the MICOS complex include two subcomplexes: MIC60/MIC25/MIC19 and MIC10/MIC26/MIC27 which are Rabbit Polyclonal to PPP1R2 bridged by MIC13 [18,19] using its conserved GxxxG and WN motifs [20]. The MICOS complex present in the IM is in turn connected via MIC60 to SAM50 present in the OM through MIC19 contributing to the formation of a 2 mega Dalton MIB (Mitochondrial intermembrane space bridging) complex [21,22]. Thus, the MIB complex mediates the IM and OM contacts. The MICOS complex correlates with the presence of cristae. In fact, a very recent study showed that Mic60 is involved in the development of intracytoplasmic membranes in alphaproteobacteria [23]. Mic60 is conserved from alphaproteobacteria to virtually all eukaryotes, whereas Mic10 is widely distributed in the later domain of life [24,25]. SU 5416 (Semaxinib) However, there is a difference w.r.t Mic10 and Mic60 in organisms which are opisthokonts and not, likeTrypansoma brucei. TbMic60 lacks a conserved mitofilin domain while there are two TbMic10 proteins inconsistent with opisthokonts [26]. Other MICOS proteins display inconsistent phylogenetic distribution. For instance, MIC26 and MIC27 are paralogs which are only present in opisthokonts [24,25]. The MICOS complex proteins play a wide range of roles in bending the IM, formation of contact sites, protein import, mtDNA organisation, lipid metabolism, cristae dynamics, mitochondrial motility, integrity of the respiratory chain super complexes and the F1FO-ATP synthase [3,17,2734]. Consequently, it is well established that proteins of the MICOS complex are associated with various pathologies [35,36]. Mutations inMIC60were shown to be present in Parkinsons disease patients [37]. A recent report showed that homozygous mutations inMIC60led to.