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oai:figshare.com:article/34025464 2026-09-29T17:22:27Z category_4 category_8 category_12 category_13 category_24 category_272 category_734 portal_5 item_type_1 month_year_09_2026

<p>Complexome profiles of mETC complexes.</p> Andrew E. Maclean (10219517) Orsola Iorillo (22772687) Drahomíra Faktorová (373414) Monika Singh (593097) Suzanne McGill (11916067) Julius Lukeš (122896) Lilach Sheiner (79023) Biochemistry Microbiology Cell Biology Genetics Evolutionary Biology Environmental Sciences not elsewhere classified Biological Sciences not elsewhere classified shares new components mitoribosome possesses many div >< p highly conserved feature perkinsus marinus </ divergent subunit composition divergent mitochondrial complexes perkinsus </ conserved across toxoplasma </ plasmodium </ mitochondrial complexes metc complexes mitochondrial ribosome mitochondrial function xlink "> wider lineages unexpected diversity transcription factors study reveals outer membrane observed enlarged myzozoan parasite metc ), including members deadly apicomplexan complexome profiling cell survival atp synthase apiap2 family <p>(A) Immunoblot analysis of total cell lysate (Total) and purification fractions using antibodies against marker proteins of various cell compartments. The pellet from the 3,500 x <i>g</i> centrifugation step contains unbroken cells and heavy cell components, from the 40,000 x <i>g</i> step the supernatant contains soluble cytosolic proteins, and the pellet mitochondrial and organellar material. 7.5 μg of protein was loaded per lane with equal loading confirmed by instant blue staining (bottom panel). The following antibodies were used: ATPβ (mitochondrion), BiP (ER), GAPDH (cytosol) and Histone H3 (nucleus). (B) Blue-native PAGE analysis of mitochondrially-enriched material treated with βDDM and separated on a 4–16% Bis-Tris gel. Total protein (TP) was visualised by Coomassie Brilliant Blue staining (CBB), complex II (CII) activity by an in-gel succinate: nitroblue tetrazolium (NBT) activity stain, complex IV (CIV) activity by an in-gel cytochrome <i>c</i>: 3,3′-Diaminobenzidine (DAB) activity stain and ATP synthase (AS) by immunoblot analysis using the anti-ATPβ antibody. (C) Heatmaps of the complexome profiles from mitochondrially-enriched material of mETC complex subunits from complex II, complex III and complex IV. Each row represents a gel slice, with the gel slice number indicated on the left and the molecular weight, based on the migration of mammalian mitochondrial complexes (black) and soluble proteins (blue), on the right. Subunit IDs are shown at the bottom of their corresponding profile, with mitochondrially-encoded subunits highlighted in red. Dark blue indicates the highest relative abundance (1) and white the lowest (0). Coomassie-stained Native PAGE gel on which the analysis was performed is depicted on the left of the profile. Data shown is from replicate 1. (D) Coulson plots depicting the composition of mETC complexes in key species from myzozoans and ciliates. <i>Perkinsus</i> subunits were used for homology searches using BLAST. <i>T.t Tetrahymena thermophila</i>; <i>P.t Paramecium tetraurelia</i>; <i>T.g Toxoplasma gondii</i>; <i>P.f Plasmodium falciparum</i>; <i>C.p Cryptosporidium parvum</i>; <i>C.m Cryptosporidium muris</i>; <i>C.v Chromera velia</i>; <i>V.b Vitrella brassicaformis</i>; <i>P.g Polarella glacialis</i>; <i>S.m Symbiodinium microadriaticum</i>. Full dataset in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1014543#ppat.1014543.s011" target="_blank">S6 Table</a>. (E) Abundance profile of complex II subunit SDHG, complex III subunit RIP1 and complex IV subunit Cox2B across all gel slices. Protein abundance is displayed as iBAQ values. Data shown is from whole-cell complexome. (F) Abundance profile of ISPR1, ISPR2 and complex III subunit RIP1 across all slices. Protein abundance is displayed as iBAQ values. Data shown is from replicate 2. (G) Immunofluorescence assay of <i>Tg</i>ISPR. Samples labelled with anti-Ty to detect <i>Tg</i>ISPR-Ty (cyan) and anti-Tom40 (magenta) to detect the mitochondrial marker protein Tom40. Scale bar is 2 µm. Plot to the right displays fluorescence profiles of Ty (cyan) and Tom40 (magenta) labelling along the white dotted line on the merged image.</p> 2026-09-29T17:22:05Z Image Figure 10.1371/journal.ppat.1014543.g002 https://figshare.com/articles/figure/_p_Complexome_profiles_of_mETC_complexes_p_/34025464 CC BY 4.0