ABSTRACT
The capacity of mitochondria to produce ATP is linked with its ability to generate phosphatidylethanolamine (PE). Of the four separate PE biosynthetic pathways in eukaryotes, three reside in the endoplasmic reticulum (ER) and one occurs in the inner membrane (IM) of mitochondria and is executed by phosphatidylserine (PS) decarboxylase (Psd1p). Deletion of Psd1 is lethal in mice and disrupts mitochondrial morphology, impairs cell growth, and diminishes respiratory capacity in both yeast and mammalian cells. This implies that the ER pathways for PE biosynthesis are unable to compensate for the absence of Psd1. We hypothesize that this failure is due to the inefficient trafficking of extra-mitochondrial PE into the IM. Here, we sought to determine which aqueous compartment, the cytosol or the mitochondrial intermembrane space, is the greatest barrier for PE trafficking to the IM. Yeast strains expressing two novel chimeric constructs that re-direct Psd1p to either the outer mitochondrial membrane (OM-Psd1p) or the endomembrane system (ER-Psd1p) were characterized. By comparing growth phenotypes, phospholipid turnover, and the activities of electron transport chain complexes of these PE re-routed strains, we identified the intermembrane space as the greatest barrier for PE import. Interestingly, PE made outside of the IM was able to rescue the activity of respiratory complex IV but not complex III. Since the activity of both complexes is dependent on PE, this suggests that Psd1p and/or PE made in the IM is uniquely able to support complex III function.
- ABBREVIATIONS
- CL
- Cardiolipin
- complex III
- Cytochrome bc1 complex
- complex IV
- cytochrome oxidase
- CoQ
- Coenzyme Q
- DAG
- diacylglycerol
- ER
- endoplasmic reticulum
- IM
- inner membrane
- IMS
- intermembrane space
- CCCP
- m-chlorophenyl hydrazine
- MAM
- mitochondrial-associated membrane
- MICOS
- mitochondrial contact site and cristae organizing system
- mtDNA
- mitochondrial DNA
- DDM
- n-Dodecyl-β-D-maltoside
- OM
- outer membrane
- OXPHOS
- oxidative phosphorylation
- PA
- phosphatidic acid
- PC
- phosphatidylcholine
- PE
- phosphatidylethanolamine
- PI
- phosphatidylinositol
- PS
- phosphatidylserine
- Psd1
- phosphatidylserine decarboxylase 1
- RCR
- respiratory control ratio
- SCD
- synthetic complete dextrose
- SC-LAC
- synthetic complete lactate
- SCEG
- synthetic complete ethanol glycerol
- TMPD
- tetramethyl-p-phenyldiamine
- WT
- wildtype