Mitochondrial complexome reveals quality-control pathways of protein import - Nature.com

Several functions have been assigned to Rsp5, including regulation of biosynthesis of unsaturated fatty acids, ubiquitylation of endoplasmic reticulum–mitochondria contact sites, and the fusion GTPase Fzo1, protein degradation in the cytosol and multivesicular body formation 29, 30, 31, 32.

Tagging and deletion of open reading frames were performed by homologous recombination using DNA cassettes amplified by PCR using Taq and Vent polymerase (NEB) 44 or KOD hot-start DNA-Polymerase (Merck Millipore).

Arushi Gupta, Jiyao Song, Christoph Mårtensson, Lars Ellenrieder, Chantal Priesnitz, Sebastian P. Straub, Kim Nguyen Doan, Bogusz Kulawiak, Heike Rampelt, Nils Wiedemann & Nikolaus Pfanner

Quality and usability of MitCOM benefitted particularly from the high-resolution sampling of the blue native gel, the advanced processing of MS-data for determination of accurate protein abundance–mass profiles and the new viewer toolbox including multi-Gaussian component fitting and correlation analysis.

Top insets are stepwise zooms into the plot resolving a closely co-localized group of protein profile segments representing the main peak of succinate dehydrogenase (respiratory chain complex II) assembled from four subunits Sdh1–4.

WT yeast cells expressing the standard precursor cytochrome b 2-DHFR LPxY or the mutant form b 2-DHFR GGxY and His-tagged ubiquitin as indicated were lysed under denaturing conditions and subjected to affinity purification via Ni-NTA.