Phosphatidylethanolamine made in the inner mitochondrial membrane is essential for yeast cytochrome bc1 complex function

Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III.


Trp1 Forward
Used for PCR screens for Trp1based PCR disruptions.
Morphology of purified complex IV SMALP Supplementary Fig 8. Cox8-His is functional, assembles normally, and enables affinity purification of complex IV. (A) Cox8-His was detected in yeast whole cell extracts of the indicated strains by immunoblot. Pic1 served as a loading control. (B) The indicated strains were spotted and incubated at 30°C for 2 days on YPD and for 3 days on rich lactate (RL). (C) Blue native-PAGE analysis of respiratory supercomplexes (RSCs) using mitochondrial extracts solubilized in 1.5% (w/v) digitonin. Complex III assembly was monitored by immunoblot against the nuclear-encoded subunit Rip1 and complex IV assembly was monitored by immunoblot against the nuclear-encoded subunit Cox4. Mitochondria lacking CL (crd1Δ) were used as a positive control for RSC destabilization. (D) SMA extracts from WT yeast lacking or expressing Cox8-His were affinity purified using Ni-NTA agarose, washed sequentially with wash buffer containing 10 mM and 20 mM imidazole, and bound material eluted with 60 mM imidazole. Equal amount of the indicated fractions were resolved by SDS-PAGE and immunoblotted for complex IV subunits (His, Cox2, and Cox3), two abundant OM proteins (OM45 and Tom70), and Kgd1. (E) Electron micrograph of affinity purified CIV-SMALPs at a magnification of 98,000x (scale bar = 100 nm). (F) Mitochondria from the indicated strains were immunoblotted as designated.   Supplementary Fig 10. Cellular and mitochondrial phospholipid profiles from choline (+C) and ethanolamine (+E) supplemented psd1psd2 yeast. (A-D) Cellular and (E-H) mitochondrial phospholipids from the indicated strains were labeled overnight with 32 Pi and separated by TLC. All graphs show the mean ± S.E.M. for n=6 biological replicates. Significant differences (ns, P > 0.05; 1 symbol P ≤ 0.05; 2 symbols P ≤ 0.01; 3 symbols P ≤ 0.001; 4 symbols P ≤ 0.0001) versus WT (asterisk) or psd1psd2 (number sign) were determined by one-way analysis of variance (ANOVA) with Tukey's pairwise comparison.  Fig 11. Specificity of Abf2-specific antisera. 25 g of isolated mitochondria from the indicated yeast strains were immunoblotted using antisera raised againts His 6 Abf2.Aco1p served as a loading control.
Notes for raw files of western blots: .ai Type size: 5pt .psd box size: 0.75pt Inserted jpeg files of .psd files place in .ai  Exposures were adjusted after cropping of each red boxed blot.
Note: Edge of Psd1 blot was cut with razor and resulted in "line" present between the Psd1 protein band in the ER-Psd1 deoxycholate minus protease sample.
Data not shown in Figure 2D: Aac2p (IM) protease exposure blots; psd1psd2 subcellular fractionation (left panel) Autoexposure function on LiCOR was used to image the indicated immunoblots (white background).
Individual blot exposures were adjusted after cropping of each red boxed blot. Only red boxed blots were used in representative image. Note: exposed image does not reveal molecular weight markers; protein molecular weights were estimated from molecular weight color standard marked on nitrocellulose membrane