Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion

Abstract

Mitochondrial membrane fusion is a process essential for the maintenance of the structural integrity of the organelle. Since mitochondria are bounded by a double membrane, they face the challenge of fusing four membranes in a coordinated manner. We provide evidence that this is achieved by coupling of the mitochondrial outer and inner membranes by the mitochondrial fusion machinery. Fzo1, the first known mediator of mitochondrial fusion, spans the outer membrane twice, exposing a short loop to the intermembrane space. The presence of the intermembrane space segment is required for the localization of Fzo1 in sites of tight contact between the mitochondrial outer and inner membranes. Mutations in the intermembrane space domain of yeast Fzo1 relieve the association with the inner membrane. This results in a loss of function of the protein in vivo. We propose that the mitochondrial fusion machinery forms membrane contact sites that mediate mitochondrial fusion. A fusion machinery that is in contact with both mitochondrial membranes appears to be functionally important for coordinated fusion of four mitochondrial membranes.

Figure 4

Insertion of a linker between the transmembrane segments of Fzo1 relieves its association with the inner membrane. (A) A mutant version of Fzo1, Fzo1-2, was constructed by insertion of a linker sequence between the transmembrane segments (TM). Depicted is the topology of the mutant protein and the amino acid sequence of the intermembrane space segment. (B) Topology of the Fzo1-2 protein. Mitochondria harboring the Fzo1-2 mutant protein were analyzed as in the legend to Fig. 1 B. (C) Complex assembly of the Fzo1-2 protein. A Triton X-100 extract of fzo1-2 mitochondria was loaded on a Superose-6 column. Fractions were collected and analyzed by immunoblotting and densitometry scanning. The elution peaks and molecular masses of marker proteins are indicated (Hsp60, 850 kD; apoferritin, 440 kD; ADH, 150 kD). (D) Submitochondrial localization of the Fzo1-2 protein. fzo1-2 mitochondria were subfractionated and analyzed as in the legend to Fig. 3 A.

Figure 1

The COOH-terminal end of Fzo1 is exposed to the cytosol. (A) Members of the Fzo1 protein family contain two closely neighboring putative transmembrane segments. The hydrophobicity profile of the Fzo1 proteins of Saccharomyces cerevisiae (Sc) (available from GenBank/EMBL/DDBJ under accession no. Z36048), Schizosaccharomyces pombe (Sp) (CAA19004), Caenorhabditis elegans (Ce) (U29244, ORF14), and Drosophila melanogaster (Dm) (Hales and Fuller 1997) were plotted according to Kyte and Doolittle 1982. Hydrophobic regions are depicted in black. (B) Subfractionation and protease treatment of wild-type mitochondria. Mitochondria (M), mitoplasts (MP), or mitochondria solubilized with Triton X-100 (TX) were treated with the indicated amounts of proteinase K (PK). Then proteins were precipitated with TCA and analyzed by SDS-PAGE and immunoblotting. Fzo1 and COOH-terminal Fzo1 fragments were detected using an antiserum directed against the COOH-terminal 12 amino acid residues. Markers: D-lactate dehydrogenase (DLD), an integral protein of the inner membrane that exposes its major part to the intermembrane space; cytochrome c peroxidase (CCPO), a soluble protein of the intermembrane space; and Mge1, a soluble matrix protein. (C) Carbonate extraction of COOH-terminal Fzo1 fragments. Mitoplasts generated from strain YBW114 were treated with PK and extracted with 0.1 M Na₂CO₃. Then, membranes were floated in a sucrose gradient. Proteins were harvested from the gradient, precipitated with TCA, and analyzed by immunoblotting. Lane 1, floated membranes; lane 2, middle fraction; lane 3, bottom fraction containing soluble proteins; lane 4, pellet fraction. Markers: AAC, an integral protein of the inner membrane; and Mge1. (D) Digitonin fractionation of mitochondria. Isolated mitochondria of strain YBW114 were treated with the indicated concentrations of digitonin and then incubated in the absence or presence of 500 μg/ml PK. Mitochondria were reisolated by centrifugation and analyzed by immunoblotting as described for A. (E) Topology of Fzo1 and generation of COOH-terminal fragments. Left, topology of Fzo1 in the outer membrane (OM) and generation of the COOH-terminal 19-kD fragment by PK in intact mitochondria. Protease cleavage is indicated by an arrow. Right, generation of the 16-kD fragment when the intermembrane space (IMS) is accessible to protease. Two protease cleavage sites are indicated by arrows.

Figure 2

Import of Fzo(600–810)–DHFR and Fzo(600–810) in vitro. (A) Topology of Fzo(600–810)–DHFR in the outer membrane (OM). The distribution of methionine residues in the fusion protein is symbolized by asterisks. The methionines functioning as translation starts are numbered. N, NH₂ terminus of the fusion protein. (B) In vitro translation and import of Fzo(600–810)–DHFR. Fzo(600–810)–DHFR was synthesized in reticulocyte lysate in the presence of [³⁵S]methionine (lane 1, Lysate). For in vitro import, the protein was incubated with isolated mitochondria and the organelles were reisolated by centrifugation. Equal aliquots were either left untreated on ice (lane 2, total [T]), treated with proteinase K (lane 3, PK) or trypsin (lane 4, Try.), or were extracted with carbonate (CO₃ ²−) and separated into pellet (lane 5, P) and supernatant (lane 6, S) fractions. All samples were precipitated with TCA and analyzed by SDS-PAGE and autoradiography. The numbering of the different translation products corresponds to the numbers indicated in A. (C) Localization of the DHFR domain of imported Fzo(600–810)–DHFR. Import was performed as in B, and mitochondria were treated with the indicated concentrations of trypsin. Mitochondria were then sedimented by centrifugation, and pellet (P) and supernatant (S) fractions were precipitated with TCA and analyzed by SDS-PAGE and autoradiography. The size of the DHFR domain is indicated. The intactness of the outer membrane was controlled by immunoblotting using antiserum against cytochrome c peroxidase (CCPO). (D) Import of Fzo(600–810) was performed as described for B.

Figure 3

Localization of Fzo1 in contact sites. (A) Association of Fzo1 with the inner membrane. Mitochondrial membrane fragments were generated and separated on a sucrose gradient as described in Materials and Methods. Proteins from fractions 4–10 were precipitated with TCA and analyzed by Western blotting. Porin was used as a marker for the outer membrane, and AAC was used as a marker for the inner membrane. (B) Release of Fzo1 from the inner membrane by aqueous perturbant. Mitochondrial membrane fragments were generated in the presence of 2 M urea and analyzed as in A. (C) Localization of COOH-terminal Fzo1 fragments. Mitochondrial membrane fragments were prepared in the presence of 100 μg/ml proteinase K during the swelling step and analyzed as in A. (D) Location of Fzo1 and COOH-terminal fragments in membrane contact sites. OM, outer membrane; IM, inner membrane; and X, unknown component in the inner membrane.

Figure 5

Mutation of the intermembrane space segment compromises the function of Fzo1. (A) Growth phenotype of the fzo1-2 mutant. The fzo1-2 mutant, the Δfzo1 deletion strain, and the isogenic wild type (WT) were grown overnight in liquid minimal medium selective for the maintenance of the plasmid encoding the mutant allele (SD; 2% glucose). Then, 10-fold serial dilutions were spotted onto YPD plates (2% glucose) and YPG plates (3% glycerol). YPD plates were incubated for 3 d at 30°C; YPG plates were incubated for 4 d at 30°C. (B) Mitochondrial morphology of the fzo1-2 mutant. The fzo1-2 mutant, the Δfzo1 strain, and the isogenic wild-type expressing mtGFP were grown overnight in galactose-containing liquid minimal medium (SGal; 2% galactose), selective for maintenance of the plasmid encoding the mutant allele. Living cells were subjected to fluorescence microscopy. Left, the mitochondrial morphology of representative cells is shown; and right, the corresponding phase–contrast images are shown. (C) Quantification of mitochondrial morphology in fzo1 mutants. The following strains were grown to mid-logarithmic growth phase in liquid minimal medium under selection for the plasmids: wild-type (WT; YBW89), Δfzo1 (YBW113), fzo1-2 (YBW117), an Fzo1-2–overexpressing strain (fzo1-2[2μ]; YBW183), and an Fzo1-2–overexpressing strain complemented with a single copy FZO1 wild-type gene (fzo1-2[2μ]FZO1; YBW210). More than 100 cells per culture were examined by fluorescence microscopy and grouped into the following phenotypic classes: wild-type like (mitochondrial reticulum below the cell cortex), some tubules (mostly fragmented mitochondria with a few tubular structures present), aggregated (clustered mitochondrial fragments), and fragmented (evenly distributed mitochondrial fragments). Bar, 2 μm.

Figure 6

Hypothetical model of mitochondrial fusion mediated by a fusion machinery located in contact sites. Coordinated fusion of mitochondria might be achieved by a mechanism which requires tight contact between the outer membranes (OM) and inner membranes (IM) (see text for details). The fusion complex in the outer membrane is depicted in black; and putative interaction partners in the inner membrane are indicated by white boxes.