Fig. 2: Por1(KE) mutant is defective in Tom22 binding and impairs the MIApathway import. | Nature Communications

Fig. 2: Por1(KE) mutant is defective in Tom22 binding and impairs the MIApathway import.

From: Oligomer-based functions of mitochondrial porin

Fig. 2

a por1Δ cells expressing Por1 and mutant Por1 with the indicated mutations, or without expression (vector), were grown in SCD for at 30 °C. Cell extracts were prepared, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Pgk1, yeast phosphoglycerate kinase 1. b Serial dilutions of the por1Δ cells expressing Por1 and Por1 with the indicated mutations, or without expression (vector), were spotted on SCD and SCLac media and grown for 2 (SCD) or 3 (SCLac) days at 30 °C and 37 °C. c In this NADH transport assay, oxidation of externally added NADH was measured for the indicated reaction times for the indicated mitochondria (por1Δ cells expressing Por1 (WT) and mutant Por1 with the indicated mutations, or without expression (vector), which were isolated from cells cultured in YPD. Values are means ± SEMs (n = 3). d In vivo photocrosslinking of benzoylphenylalanine (BPA) at residue 118 of Tom22 in the indicated strains (por1Δ cells expressing WT Por1-HA and Por1(KE)-HA) was detected by anti-Tom22 antibodies (left) and anti-HA antibodies (right). 22, Tom22; 22-Por1HA, Tom22 crosslinked with Por1-HA. e Proteins in mitochondria isolated from the indicated strains were analyzed with the indicated antibodies. f, g The indicated mitochondria (WT, por1Δ, and por1Δ expressing Por1(KE)), isolated from cells grown in YPD, were incubated with the indicated radiolabeled precursors for the indicated times at 25 °C. Tim9 and Tim10 are IMS-localized MIA-pathway substrates, and Su9-DHFR and Hsp60 are matrix-localized TIM23-pathway substrates. Then the mitochondria were treated with 50 μg/mL proteinase K for 15 min on ice (for Su9-DHFR and Hsp60) or diluted 10-fold with ice-cold buffer containing 50 mM iodoacetamide and 50 μg/mL proteinase K for 15 min on ice (for Tim9 and Tim10), and PMSF was added to 1 mM. The proteins were analyzed by SDS-PAGE and radioimaging; quantification was plotted against time (bottom), where the maximum amount for WT mitochondria was set to 100%. Values are means ± SEMs (n = 3). C, 10% of the input. Source data are provided as a Source Data file.

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