Fig. 4.

Integrating the landscape of submitochondrial protein distribution. a Experimental design for global allocation of integral outer and inner membrane proteins. Highly purified mitochondria from a light yeast culture were used for isolation of highly pure outer membrane fractions³¹ (OM_L). Total membrane fractions were generated from mitochondria of a heavy labeled yeast culture (TOT_H). Both samples were mixed and the ratios OM_L/TOT_H measured by LC-MS. b OM_L/TOT_H ratios of selected integral outer and inner membrane proteins and validation by immunoblotting. c Array of determined OM_L/TOT_H ratios reveals distribution of integral mitochondrial outer and inner membrane proteins (shown as relative intensities for proteins in the OM fraction). d Sublocalization of Prd1 to the mitochondrial matrix. e Cell-free translated Prd1 and Prd1^E502Q protein was incubated with Cox4 presequence peptide (Cox4^preseq.; left panels) or radiolabeled Cox4 precursor protein (right panels) for indicated time. Ctrl., control (mock translation). f Complete overview of the landscape of submitochondrial protein distribution. Allocation of the mitochondrial proteome to the various subcompartements deciphered in this study