Journal: bioRxiv

Article Title: MTCH2 is a mitochondrial outer membrane protein insertase

doi: 10.1101/2022.09.15.508165

Figure Lengend Snippet: (A) Label-free mass spectrometry analysis of purified mitochondria isolated from K562 cells using a percoll gradient expressing a MTCH2 targeting sgRNA (kd) compared to a non-targeting control (nt). Displayed are proteins that across four biological replicates were statistically altered in MTCH2 depleted versus non-targeting guide expressing cells. (B) Western blotting of endogenous proteins from MTCH2 depleted (kd) and control cells in (generated as in A; left), and wild type and MTCH2 knock out (KO) cells (right). Substrates are colored by topology based on the key shown in (B). (C) Flow cytometry analysis of integration of outer membrane protein reporters using the split GFP system described in . GFP fluorescence relative to an RFP expression control are displayed as histograms in MTCH2 knockdown versus non-targeting K562 CRISPRi cells. Displayed are representative examples of a TA, SA, and multipass membrane protein that have a MTCH2 dependent biogenesis defect. (D) Summary of dependence on MTCH2 for the indicated outer membrane substrates determined using the fluorescent reporter system shown in (C) and colored by topology based on the key in (B).

Article Snippet: In many instances, we used a programmed dual sgRNA guide vector (( ); Addgene #140096) to allow for multiple genes to be depleted at once or to increase efficiency of knock-down.

Techniques: Mass Spectrometry, Purification, Isolation, Expressing, Control, Western Blot, Generated, Knock-Out, Flow Cytometry, Membrane, Fluorescence, Knockdown