Journal: Nature Communications

Article Title: Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis

doi: 10.1038/s41467-019-11047-6

Figure Lengend Snippet: Phosphorylation of Timm50 decreases mitochondrial protein import. a Protein domain schematic of mouse Timm50 (top) and yeast Tim50p (bottom) with highlighted phosphoresidues (red). b Western blot of overexpressed FLAG-tagged Tim50p WT, S104A, or S104E shows that mutations do not destabilize proteins. c Quantification of western blot for Tom70p protein levels (see Supplementary Figure for corresponding western blot data). d Import assays in Δ tim50 yeast mitochondria overexpressing wild type (WT) TIM50 or S104 mutants using generic import cargo cytochrome b -(167) ∆19 -DHFR. Imported proteins result in accumulation of the mature (m) versus precursor (p) or intermediate (i) processed bands. Quantification of import rates is shown over time ( e ) and as maximal import signal ( f ) (AU densitometry at 10 min). g Import assays in mitochondria isolated from WT, Δ ptc7 , or Δ ptc7 overexpressing TIM50 S104A yeast using the matrix-targeted model substrate cytochrome b -(167) ∆19 -DHFR. Imported proteins result in accumulation of the mature (m) product. Quantification of import rates is shown over time ( h ) and as maximal import signal ( i ) (AU densitometry at 10 min). j Quantification of mitochondrial proteins in mouse heart (left) ( n = 145 for matrix proteins and n = 16 for OMM proteins) and mouse liver (right) ( n = 151 for matrix proteins and n = 21 for OMM proteins) showing significantly lower expression (*** = p < 0.001, as calculated by a two-tailed Student’s t -test) of candidate Timm50 substrates (e.g., MTS and matrix-containing proteins) than proteins that localize to the outer mitochondrial membrane (OMM). For box plots in j , center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, and outliers are represented by white dots. k , l Volcano plots of candidate Timm50 substrates in heart ( k ) and liver ( l ) show downregulation of metabolic proteins in amino acid metabolism and fatty acid oxidation, both of which are disrupted in Pptc7-null tissues (Fig. ). Source data for panels b , d , g , and j – l are provided as a Source Data file

Article Snippet: Recombinant Mdh1p, Atp2p, and cytochrome b 2 -(167) ∆19 -DHFR were generated using the quick TnT® Quick Coupled Transcription/Translation System (Promega) supplemented with 35 S-Methionine and Cysteine (EasyTag EXPRESS35S protein labeling mix, Perkin Elmer) according to the manufacturer’s instructions.

Techniques: Western Blot, Isolation, Expressing, Two Tailed Test, Software