Proteinase K

1. Fig S1 Cytosol Mitochondria - + + Proteinase K - - + Triton X-100 mtPARN ~74 kDa mtPARNC ~53 kDa ~70 kDa mtLUC 1 2 3 4 Figure S1 Derivatives of poly(A) specific 3’ exoribonuclease, PARN, are successfully targeted to mitochondria. FLAG tagged full length or C-terminal truncated PARN carrying a mitochondrial presequence were expressed from transfected HEK293 cells. Mitochondria were prepared (lane 2), treated with Proteinase K alone (lane 3), or lysed with Triton X-100 and treated with proteinase K and separated by SDS PAGE alongside post mitochondrial supernatant (lane 1, cytosol). Western blotting with antibodies to the FLAG moiety indicate mitochondrial localisation and internalisation.

2. Fig S2 A Respiration [pmol O2/(s*million of cells] mtPABP1 B mtLUC mtPABP1 mtLUC ~70 kDa MRPL3 ~38 kDa ~28 kDa MRPS18B Figure S2 A. High resolution respirometry was performed on transfectants expressing mtPABP1 () or mtLUC () for 8 days in glucose medium. Rates of oxygen consumption at rest and following the sequential addition of oligomycin, FCCP (uncoupled), rotenone and antimycin were performed as detailed in Supplemental Methods. Parameters describing the relative coupling efficiencies between respiration and oxidative phosphorylation are defined in Supplemental Methods. B. mtPABP specifically immunoprecipitates intact mitoribosomes. Immunoprecipitation via the FLAG tag was performed on HEK293 cells expressing either mtLUC-FLAG or mtPABP-FLAG as described in the materials and methods. Equal volumes of immunoprecipitated eluate were separated by SDS PAGE and westerns performed. Antibodies against FLAG confirmed successful immunoprecipitation and interrogated the eluate for the large 39S (MRPL3) and small 28S (MRPS18B) subunits of the human mitochondrial ribosome.