Microproteins represent a class of short polypeptides with very diverse cellular functions. These small proteins frequently escape proteomic-based identifications, making their extent and theeir potential functions largely elusive. Many microproteins originate from transcripts that are annotated as non-coding such as long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and micro RNAs (miRNAs). However, also messenger RNAs (mRNAs) can contain additional short open reading frames (sORFs) that encode microproteins. In this work, we functionally characterize SMIM26, a microprotein localized to mitochondria. In biochemical and single molecule tracking studies, we find that SMIM26 interacts with VDAC1/2 in the outer mitochondrial membrane and SLC25A6 in the inner mitochondrial membrane. We show that it spans the intermembrane space where it interacts with PGAM5 and is phosphorylated at distinct residues. Knockout cancer cell lines are viable but have strongly reduced respiratory chain activity and are sensible to glucose starvation. Interestingly, knockout mice are not viable and die already at early developmental stages. Zebrafish homozygous smim26 mutants are viable, but show reduced fitness and survival compared to their wild-type or heterozygous siblings. Surprisingly, loss of smim26 induces female-to-male sex reversal in mutant fish. Consistent with the mitochondrial phenotype in cell lines, respiration is reduced in homozygous zebrafish embryos and components of the N- and Q-module in respiratory chain complex I are downregulated. In cancer, loss of SMIM26 leads to dysregulated signaling pathways and high expression levels of SMIM26 alone or in combination with its binding partners is associated with worse survival in liver cancer. Based on the functional characterization and patient data, in a pilot anti-cancer drug screen we identify two compounds that display a stronger effect SMIM26 on knockout cell lines compared to wild-type. Our work suggests that SMIM26 coordinates metabolite transport through the inner and outer mitochondrial membranes and is essential for respiratory function in vivo. Therefore, SMIM26 could be an interesting target for anti-cancer therapies in the future.
Kevin Michael Heizler (Thu,) studied this question.