Choline supplementation rescues reduced Cnm1-GFP levels in strains lacking genes related to PC biosynthesis. (A) Cells overexpressing Cnm1-GFP (OE Cnm1-GFP) on the background of deletions in cho2, opi3, and ino2 were grown to mid-logarithmic phase in synthetic minimal medium and imaged with or without 5 mM choline. Mitochondria were dyed using MitoTracker Orange. Scale bar, 5 µm. (B) Cells lacking ino4 and overexpressing (OE) Cnm1-GFP were grown to mid-logarithmic phase in synthetic minimal medium and imaged with or without 5 mM choline supplementation. Mitochondria were stained using MitoTracker Orange. Scale bar, 5 µm. (C) Quantitation of the overexpressed (by TEF2pr) Cnm1-GFP signal brightness in either control or Δopi1 strains, determined by the mean intensity level of the 488-nm excitation wavelength using ScanR Olympus soft imaging solutions, version 3.2. While the mean intensity was maintained in most control cells, deletion of opi1 resulted in a higher probability of having cells with stronger Cnm1-GFP signal. a.u., arbitrary units. (D) An example of the strains quantified in C. Overexpression of Cnm1 tagged with GFP (OE Cnm1-GFP) on its C terminus on the background of opi1 deletion showed enhanced GFP signal intensity in some of the cells compared with control. Mitochondria were dyed using MitoTracker Orange. Scale bar, 5 µm. (E) Cells overexpressing (OE) Cnm1 and C terminally tagged with mCherry on the background of Δcho2 strain were grown to mid-logarithmic phase in synthetic minimal medium and imaged with or without supplementation of 1 mM PC. Scale bar, 5 µm.