Domain architecture of Cnm1 and the effect of losing its TMD on mitochondrial morphology. (A) Prediction of an internal mitochondrial targeting signal–like (iMTS-L) sequence in Cnm1 calculated as described before (Boos et al., 2019). A peak with the highest TargetP1 scores can be found around amino acids 350–370 of the nuclear protein Cnm1, suggesting the presence of an iMTS-L sequence in this region. Since iMTS-Ls have been shown to directly bind Tom70, this highlights this region as a potential binding interface of Cnm1 with Tom70 on the mitochondrial membrane. (B) An illustrated model of Cnm1 protein containing the localization of its two predicted transmembrane domains and the predicted iMTS-like signals. (C) Overexpression of the soluble Cnm1 (Δ1–112 aa) tagged with GFP on its N terminus (OE GFP-Δtmd-Cnm1) has a dramatic effect on mitochondrial morphology. The ER is marked by a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL). Mitochondria were dyed with MitoTracker Orange. Scale bar, 5 µm.