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Figure 2


Fig. 2. Protection of Fis1-TMC from modification with IASD represents proper insertion into the outer membrane. (A) The sequences of the tail domains of native Fis1 and its variants. Only relevant residues in the cytosolic domain are indicated. The putative transmembrane segment is underlined. Cysteine and positively charged residues in the wild-type sequence and their replacement in the variant proteins are in bold type. (B) Fis1-TMC can complement the mitochondrial morphology phenotype of the fis1{Delta} strain. Cells of the indicated strains (containing mitochondria-targeted RFP) were analyzed by fluorescence (left) and phase-contrast (right) microscopy. (C) Endogenous Fis1-TMC is protected from modification with IASD. The indicated amounts of mitochondria isolated from a fis1{Delta} strain transformed with Fis1-TMC were incubated where indicated with IASD in the presence or absence of Triton X-100 (Tx-100). Mitochondrial proteins were analyzed by SDS-PAGE and immunocytochemistry with antibodies against Fis1. (D) Radiolabeled Fis1-TMC is protected from modification with IASD. Wild-type mitochondria were incubated with either radiolabeled Fis1 or radiolabeled Fis1-TMC for 30 minutes at 25°C. Mitochondria were re-isolated by centrifugation and resuspended in either import buffer or labeling buffer containing, where indicated, IASD and Tx-100. Mitochondrial proteins were analyzed by SDS-PAGE and autoradiography. (E) An insertion-deficient Fis1 variant is not protected from modification with IASD. Wild-type mitochondria were incubated with either radiolabeled Fis1-TMC or Fis1(TMC-4Q) for 30 minutes at 25°C. Further treatment was as described in D. (F) The modification of Fis1-TMC is cysteine specific. Radiolabeled precursors of Fis1(CS) and Fis1-TMC were analyzed directly by SDS-PAGE or were incubated for 20 minutes at 25°C in labeling buffer containing, where indicated, IASD and Tx-100. Further treatment was as described in D. A band representing hemoglobin, which is present at high amounts in the reticulocyte lysate is indicated with an asterisk.





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