A mutation associated with CMT2A neuropathy causes defects in Fzo1 GTP hydrolysis, ubiquitylation, and protein turnover

Abstract

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.

Figure 1.

Schematic representation of Fzo1 domain structure, including three heptad repeat regions (HR0–HR2), a GTPase domain (including conserved GTPase motifs G1–G4) and a transmembrane region (TM). Fzo1 amino acid substitutions analogous to human Mfn2 mutations found in CMT2A patients (in parentheses) are indicated. Below, the GTPase domain is expanded to show the alignment of Mfn2 and Fzo1 protein sequences in the conserved G1–G4 motifs.

Figure 2.

In vivo mitochondrial phenotypes of cells expressing mutant Fzo1 proteins. (A) Quantification of mitochondrial morphologies observed in cells expressing WT and mutant Fzo1 proteins. Bars and error bars represent the average and SD of at least three independent experiments. Samples are labeled according to the amino acid substitution in the protein being expressed. (B) Representative images of mitochondrial morphology in fzo1Δ cells expressing the indicated FZO1 gene from the pRS414 plasmid. Mitochondria were visualized by expression of mito-GFP. (C) Quantification of mitochondrial fusion in fzo1Δ dnm1Δ cells expressing WT or mutant Fzo1 protein. The percentage of large-budded zygotes containing fused mitochondria (overlapping red and green mitochondrial tubules) in each strain is indicated. Bars and error bars represent the average and SD from three independent experiments. At least 50 zygotes were scored per experiment. (D and E) Dilution analysis of respiratory growth phenotypes in fzo1Δ (D) or FZO1 (E) cells expressing the WT or mutant FZO1 gene from the pRS414 plasmid. Each spot is a serial 1:10 dilution of cells grown on SD-TRP (dextrose) or SGly-TRP (glycerol) plates at 30°C.

Figure 3.

Coimmunoprecipitation of HA and Myc-tagged Fzo1 proteins. Lysates from cells expressing the indicated HA and Myc-tagged Fzo1 proteins were incubated with anti-Myc agarose beads. Lysates (bottom) and immunoprecipitated proteins (top) were resolved by SDS-PAGE and probed by Western blotting with anti-HA and anti-Myc antibodies.

Figure 4.

An assay for GTP hydrolysis by immunoprecipitated Fzo1 protein. (A) A schematic detailing the steps for immunoprecipitation of Fzo1 protein from isolated mitochondria and the subsequent analysis of GTP hydrolysis by the immobilized protein. (B) Example of TLC separation of radiolabeled GDP from nonhydrolyzed GTP. The initial buffer containing [α-³²P]GTP was spotted in the lane 1. The conversion of GTP to GDP by WT Fzo1 protein is shown in lane 2. Three Fzo1 proteins with GTPase domain mutations (K200A, S201N, and T221A) have no GTP hydrolysis activity (lanes 3, 4, and 5). (C) WT and mutant HA-tagged Fzo1 proteins were immunoprecipitated from isolated mitochondria and incubated with [α-³²P]GTP. The average amount of [α-³²P]GDP (a measure of GTP hydrolysis) generated by each mutant protein relative to WT is shown in the graph. Bars and error bars are the average and SD from three independent experiments. (D) Mitochondria isolated from strains expressing HA and Myc-tagged WT or V327T Fzo1 protein(s) were used to IP or coIP the indicated proteins. Fzo1 protein(s) immobilized on anti-Myc agarose beads were incubated with [α-³²P]GTP and the amount of [α-³²P]GDP generated by GTP hydrolysis was measured relative to WT. Bars and error bars are the average and SD from three independent experiments.

Figure 5.

Stabilization and delayed turnover of the V327T mutant protein is associated with diminished Mdm30 interaction and reduced ubiquitylation. (A) Steady-state abundance of WT and mutant proteins expressed in fzo1Δ cells. Whole cell extracts were immunoblotted with anti-Fzo1 and anti-3PGK antibodies. The density of each Fzo1 protein band was normalized to 3PGK signal in the same sample. The average abundance of Fzo1 protein in each strain is plotted relative to the WT sample. Error bars indicate the SEM from several blots. The asterisk indicates statistical significance (p < 0.05) by t test (GraphPad Prism Software, La Jolla, CA). (B) Mdm30-Myc was coimmunoprecipitated from cell lysates (bottom) by HA-Fzo1 and HA-V327T (top). The amount of Mdm30-Myc coimmunoprecipitated relative to HA-Fzo1 protein is reduced in the HA-V327T strain. (C) HA-Fzo1 protein turnover was analyzed by ³⁵S pulse-chase metabolic labeling in WT and V327T mutant strains. Samples were collected and analyzed at 0, 30, and 60 min after the chase was initiated. The asterisk indicates a nonspecific band. The percentage of ³⁵S-Fzo1 protein remaining at each time point is plotted. Data points are the average and error bars are the SD from three independent experiments. (D) HA-Fzo1and HA-V327T proteins were immunoprecipitated from cell lysates and probed with anti-HA antibody to detect ubiquitylated forms of the tagged Fzo1 protein (HA-Fzo1-Ub, top). A lower exposure of the same blot (bottom) shows the levels of nonubiquitylated HA-Fzo1 species.

Figure 6.

Turnover and ubiquitylation of HA-V327T is restored in the presence of WT Fzo1 protein and GTPase activity does not require Mdm30. (A) Steady-state abundance of WT and mutant Fzo1 proteins in whole cell extracts from the indicated yeast strains. Protein bands were detected using a fluorescent secondary antibody followed by scanning on an Odyssey imaging system (Li-Cor Biosciences). The average intensity of each Fzo1 protein band was normalized to 3PGK signal. Bars represent the abundance of Fzo1 protein in each strain relative to the WT sample. Error bars indicate the SEM from at least three blots. (B) Steady-state abundance of HA-tagged WT and mutant proteins expressed in FZO1 cells. Whole cell extracts were immunoblotted with anti-HA and anti-3PGK antibodies. The average intensity of each HA band was normalized to 3PGK signal. Bars represent the abundance of HA-Fzo1 protein in each strain relative to the WT sample. Error bars indicate the SEM from at least three blots. (C) HA-Fzo1and HA-V327T proteins were immunoprecipitated from cell lysates and probed with anti-HA antibody to detect ubiquitylated forms of the tagged Fzo1 protein. HA-Fzo1 in mdm30Δ cells is not ubiquitylated. HA-V327T is ubiquitylated in cells that also express WT Fzo1. (D) HA-tagged Fzo1 protein was immunoprecipitated from the indicated isolated mitochondria and incubated with [α-³²P]GTP. Bars represent the mean [α-³²P]GDP generated by Fzo1 protein from each strain relative to WT. Error bars are the SD from three independent experiments.