Cnm1-mediated contact sites are affected by PC metabolism. (A) Schematic illustration of the biosynthesis pathway of PC. PS formed in the ER is transferred to mitochondria to generate PE, which is then transferred back to the ER for the formation of PC by Cho2 and Opi3. Ino2 and Ino4 are the transcriptional activators of both Cho2 and Opi3. Opi1 is a negative regulator of the pathway. PC molecules can also be synthesized through the Kennedy pathway when exogenous choline is present. IM, inner membrane. (B) Deletion of PC biosynthesis–related genes reduced Cnm1 signal levels. Overexpressed (OE) Cnm1 was tagged with GFP on its C terminus and mitochondria were stained using MitoTracker Orange. Scale bar, 5 µm. (C) Reduced levels of Cnm1-GFP (expressed from a strong constitutive promoter) in strains harboring a deletion of cho2, opi3, or ino2 can be rescued by addition of choline. Western blot analysis of four different strains without or with 5mM choline supplementation. Immunoblotting was performed with antibodies against GFP and Histone H3 as a loading control. (D) Cnm1 mediated mitochondrial clustering around the nucleus is dependent on choline levels. Cells overexpressing Cnm1 under the TEF2 promoter and harboring deletion of cho2, opi3, or ino2 were grown to mid-logarithmic phase in synthetic minimal medium without or with 5mM choline. The nucleus is visualized by Nsg1-GFP and mitochondria by MitoTracker Orange staining. Scale bar, 5 µm. (E) Overexpression of Cnm1 using the TEF2 promoter in strains deleted for proteins involved in PC biosynthesis resulted in a reduced growth rate. Strains were grown overnight in synthetic minimal medium, back diluted to OD600∼0.05 and monitored for growth over 48 h. (F) Choline buffered the growth defect of overexpressing Cnm1 in strains deleted for genes involved in PC biosynthesis. Strains were grown overnight in synthetic minimal medium, back diluted to OD600∼0.05 and monitored for growth over 48 h with or without 5mM choline supplementation.
