Structures of Fis1/Mdv1 and Fis1/Caf4 complexes. (A) Domain arrangement of Mdv1, Caf4, and Fis1. Regions A and B in Caf4, located in the N-terminal extension, directly bind Fis1, as shown in the crystal structure (C). The corresponding region B and the predicted region A in Mdv1 are also labeled. For each protein, the region within the blue frame is present in the crystallization studies. (B) Crystal structure of the Fis1/Mdv1 complex depicted in a ribbon representation. The Fis1 N-terminal arm (red) remains packed against the original hydrophobic groove and stabilizes the αB helix of Mdv1 (cyan), which packs against a second hydrophobic groove on the concave surface of Fis1. (C) Crystal structure of the Fis1/Caf4 complex. In addition to the αB helix packed against the concave Fis1 surface, Caf4 (purple) uses the αA helix and the intervening loop to bind the convex Fis1 surface. (D) The Fis1/Caf4 structure with Fis1 shown in a surface representation. The long loop allows Caf4 to straddle both sides of the Fis1 TPR domain.

The αA and αB helices are essential for adaptor function. (A) Interactions of Mdv1 αB residues with Fis1. Fis1 is shown in a surface representation with key residues highlighted. Mdv1 αB residues (cyan) are shown in a stick representation. (B) Interactions of Caf4 αA residues with Fis1. (C) Immunoprecipitation of HA-tagged Mdv1 mutants with Myc-tagged Fis1. (D) Immunoprecipitation of HA-tagged Caf4 mutants with Myc-tagged Fis1. (E) Subcellular localization of Mdv1 mutants. The localization of GFP–Mdv1 was scored by comparing to mito-DsRed in yeast carrying the indicated Mdv1 mutation. Representative examples of mitochondrial and cytoplasmic localization are shown. (F) Rescue of mdv1Δ yeast with Mdv1 mutants. Mitochondria in cells expressing the indicated Mdv1 constructs were scored with mitochondrially targeted GFP. In all phenotypic characterizations, 200 cells were scored, and percentages are shown. Representative examples of yeast with tubular and net-like mitochondria are shown.

The two binding sites on Mdv1 and Caf4 are necessary and sufficient for binding to Fis1. (A) Sequence alignment of a portion of the N-terminal extension region (N-term) of Mdv1 and Caf4. Sequences constituting binding sites αA and αB are indicated, and residues contacting Fis1 are highlighted by black triangles. Identical residues are white with a red background, and similar residues are red with a blue frame. (B) Yeast two-hybrid assay. Fis1 expressed from the pGAD vector was tested against Mdv1 or Caf4 constructs containing the N-terminal region, the C-terminal region (WD40), αA alone, αB alone, or αA and αB. Growth on adenine-deficient plates indicates an interaction.

Surface residues essential for Fis1 function. (A) Immunoprecipitation of HA-tagged Mdv1 by Myc-tagged Fis1 containing mutations in surface residues contacting the αB helix of adaptors. wt, wild type. (B) Ability of Fis1 mutants to rescue mitochondrial morphology and Dnm1 recruitment in fis1Δ yeast. Mitochondrial morphology was scored by using mito-DsRed. Subcellular localization of GFP–Dnm1 puncta was scored by comparison with mito-DsRed. (C) Schematic of fission complex recruitment by Fis1. In the unliganded state (shown at left), Fis1 resides in the mitochondrial outer membrane (OM) with its N-terminal arm (red region) packed against the concave surface of the TPR domain. The adaptors Mdv1 and Caf4 use two N-terminal helices to bind Fis1. The Fis1 N-terminal arm stabilizes adaptor binding by interacting with the αB helix (complex shown at right). The WD40 region of the adaptors then recruits Dnm1 to the mitochondria. Dnm1 exists as dimers and is activated by assembly into higher-order oligomers (not shown).