Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p - PubMed (original) (raw)

Figure 5

Fis1p is a mitochondrial membrane protein with its amino terminus exposed to the cytoplasm. (A–F) Wild-type (ADM551) cells expressing GFP-Fis1p (A–C; pRS415MET25 + rsGFP-FIS1 aa1-155) or GFP-Fis1p lacking the COOH-terminal transmembrane domain (D–F; pRS415MET25 + rsGFP-FIS1 aa1-127) were stained with MitoTracker red CMXRos (B, C, E, and F). Confocal microscopy was used to evaluate the distribution of the GFP fusion proteins (green) and mitochondrial compartments (red) in individual (A, B, D, and E) and merged (C and F) images. Bar, 5μm. (G) The distribution of Myc-Fis1p (black arrows) (ADM551; pRS415_MET25_ + 9xMYC-FIS1) was determined by immunogold labeling of ultrathin cryosections. 5-nm gold particles were found concentrated at the periphery of the mitochondrial compartment. Percentage distribution of 1,807 gold particles counted: mitochondrial tips 8.5%, mitochondrial sides (excluding constriction sites) 77.6%, mitochondrial constriction sites 12.5%, and cytoplasm 1.4%. m, mitochondria; pm, plasma membrane. Bar, 0.1 μm. (H) Total cell extracts from _fis1_Δ (lane 1, ADM549), wild-type (lane 2, ADM548), or _fis1_Δ cells expressing either Fis1p (lanes 3, 5, 7, and 9, ADM549; pRS416 + FIS1), GFP-Fis1p (lanes 4 and 6, ADM549; pRS415MET25 + rsGFP-FIS1) or Myc-Fis1p proteins (lanes 8 and 10, ADM549; pRS415MET25 + 9xMYC-FIS1) were analyzed by Western blotting with anti–Fis1p, –GFP, and –Myc serum as indicated. a, the position of GFP-Fis1p (47 kD); b, Myc-Fis1p (35 kD); and c, native Fis1p (17 kD). (I) Wild-type protein extract was fractionated by differential centrifugation, separated by SDS-PAGE, and analyzed by Western blotting with anti–Fis1p, –porin, and –3-PGK serum. Cytosol, postnuclear cytoplasmic extract; PMS, supernatant depleted of mitochondria; Mito, mitochondrial pellet. (J) Untreated (IM), osmotically shocked (OS), and Triton X-100 solubilized (TX) mitochondrial fractions were treated with 0, 10, or 100 μg/ml Trypsin and analyzed by SDS-PAGE and Western blotting with anti–GFP, –Fis1p, –cytochrome b2, and –KDH serum. *Protease-clipped form of KDH.