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. 2013 Feb;12(2):233-43.
doi: 10.1128/EC.00226-12. Epub 2012 Dec 7.

A uvs-5 strain is deficient for a mitofusin gene homologue, fzo1, involved in maintenance of long life span in Neurospora crassa

Kiminori Kurashima  1 , Michael Chae, Hirokazu Inoue, Shin Hatakeyama, Shuuitsu Tanaka
Affiliations

A uvs-5 strain is deficient for a mitofusin gene homologue, fzo1, involved in maintenance of long life span in Neurospora crassa

Kiminori Kurashima et al. Eukaryot Cell. 2013 Feb.

Abstract

Mitochondria are highly dynamic organelles that continuously fuse and divide. To maintain mitochondria, cells establish an equilibrium of fusion and fission events, which are mediated by dynamin-like GTPases. We previously showed that an mus-10 strain, a mutagen-sensitive strain of the filamentous fungus Neurospora crassa, is defective in an F-box protein that is essential for the maintenance of mitochondrial DNA (mtDNA), long life span, and mitochondrial morphology. Similarly, a uvs-5 mutant accumulates deletions within its mtDNA, has a shortened life span, and harbors fragmented mitochondria, the latter of which is indicative of an imbalance between mitochondrial fission and fusion. Since the uvs-5 mutation maps very close to the locus of fzo1, encoding a mitofusin homologue thought to mediate mitochondrial outer membrane fusion, we determined the sequence of the fzo1 gene in the uvs-5 mutant. A single amino acid substitution (Q368R) was found in the GTPase domain of the FZO1 protein. Expression of wild-type FZO1 in the uvs-5 strain rescued the mutant phenotypes, while expression of a mutant FZO1 protein did not. Moreover, when knock-in of the Q368R mutation was performed on a wild-type strain, the resulting mutant displayed phenotypes identical to those of the uvs-5 mutant. Therefore, we concluded that the previously unidentified uvs-5 gene is fzo1. Furthermore, we used immunoprecipitation analysis to show that the FZO1 protein interacts with MUS-10, which suggests that these two proteins may function together to maintain mitochondrial morphology.

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Figures

Fig 1
Fig 1
Comparison of apical growth rates and mutagen sensitivities of uvs-5 and mus-10 strains. (A) Apical growth rates. Conidia from the 1st subcultures of age-matched strains (wild-type strain, WT-bc-1; uvs-5 strain, u5-bc-1; and mus-10 strain, KTO-10H-10A) were used to inoculate race tubes. When the growth front had crossed over to the other end of the tube, some of the fresh mycelia were used to inoculate fresh race tubes. All race tubes were incubated at 25°C under constant light. Curves represent typical data from 1 of 5 independent experiments. The life span was 417.6 ± 14.6 h (n = 5) for the uvs-5 strain and 416.6 ± 10.2 h (n = 5) for the mus-10 strain. The maximum length of growth was 89.1 ± 0.5 cm (n = 5) for the uvs-5 strain and 87.6 ± 1.1 cm (n = 5) for the mus-10 strain. All data are shown in Fig. S1 in the supplemental material. (B) Spot test analysis was performed using age-matched conidia as described for panel A, along with the original FGSC uvs-5 mutant (FGSC uvs-5; strain FGSC2970) and the seventh subculture of the mus-10 strain (strain KTO-10H-10A). Ten-microliter conidial suspensions (1 × 106 conidia/ml or one of five 1:4 serial dilutions) were spotted onto agar plates containing 0.01% MMS or 0.5 mg/ml histidine, as indicated. In the case of UV, conidia were exposed to 300 J/m2 of UV after spotting. Plates were photographed after incubation for 2 days at 30°C.
Fig 2
Fig 2
Mitochondrial morphology. Growing hyphae were stained with MitoTracker Green FM (Invitrogen) and visualized using a confocal laser scanning microscope. Hyphae of the age-matched strains were generated using conidia from the first or fifth successive subculture, as shown in parentheses.
Fig 3
Fig 3
N. crassa fzo1 is a mitofusin gene homologue. (A) Genetic and physical map of the region between tyr-1 and phe-2. On the right arm of linkage group III (LGIIIR), 56 putative ORFs are presented. (B) Structures of mitofusin homologues. The lengths of the polypeptides are shown on the right. GTPase and transmembrane domains are indicated by black and gray boxes, respectively. (C) Alignment of amino acid sequences of the GTPase domains of mitofusin homologues. Identical and similar residues among proteins are represented by black and gray boxes, respectively. An asterisk shows the glutamine residue that is changed to arginine in the uvs-5 mutant. The conserved GTPase motifs, G1, G2, G3, and G4, are outlined. (D) Single base substitution detected in the fzo1 gene of the uvs-5 strain. This mutation changes the amino acid at position 368 from glutamine (Q) to arginine (R).
Fig 4
Fig 4
Effects of expressing wild-type or mutant FZO1 protein on the uvs-5 mutant. (A) Detection of FLAG-tagged FZO1 protein by Western blotting (WB). A fragment encoding FLAG-tagged FZO1 under the control of the ccg-1 gene promoter was targeted to the his-3 locus of the uvs-5 his-3 strain. One resulting transformant, uvs-5+FZO1, expressed the FLAG-tagged FZO1 protein. A vector-control transformant (uvs-5+vector) and a wild-type strain could not produce the FLAG-tagged FZO1 protein. The position of the FLAG-tagged FZO1 protein is indicated by an arrow, while an asterisk indicates a nonspecific band. (B) The mitochondrial morphologies of growing hyphae derived from conidia from the first subcultures of the indicated strains were observed using the methods described in the legend to Fig. 2. (C) Apical growth rates of strains were measured using the same procedure as that described in the legend to Fig. 1. Race tubes were originally inoculated using conidia from the first subcultures of various strains. (D) Mutagen sensitivities of strains were determined by spot test analysis as outlined in the legend to Fig. 1. Conidial suspensions were obtained using the first subcultures of the indicated strains. (E) The uvs-5+vector and uvs-5+FZO1(WT) strains are the same as those described for panel A. The uvs-5+FZO1(Q368R) strain, which expresses a mutated FZO1 protein from the ccg-1 gene promoter, was generated using procedures similar to those described for panel A, but in this case, the FLAG-tagged FZO1 protein was obtained through amplification of genomic DNA from the uvs-5 strain. Since the levels of FZO1 protein in the uvs-5+FZO1(WT) and uvs-5+FZO1(Q368R) strains were significantly different, 100 μg and 500 μg of protein, respectively, were loaded into the gel. For the vector control (uvs-5+vector), 500 μg of protein was used. The lower panel shows a longer exposure of the same blot. An arrow indicates the band corresponding to the FLAG-tagged FZO1 protein, while an asterisk shows the location of a nonspecific band.
Fig 5
Fig 5
Knock-in of the A1103G mutation into the fzo1 gene. (A) A construct carrying the A1103G mutation of fzo1 and the Bar marker was used for the knock-in procedure. The numbers indicate distances (base pairs) from the start codon of fzo1, while the asterisk marks the location of the A1103G mutation. Arrows indicate the positions of PCR primers used to amplify the DNA fragments used for sequence analysis. (B) Confirmation of Bar gene insertion into the upstream region of the fzo1 gene. The products of PCR amplification using a wild-type genome template and the primers shown in panel A were approximately 500 bp long. When the Bar gene was inserted, the size of the PCR product increased to roughly 1,900 bp. Construct, plasmid used for knock-in procedure. (C) Two bialaphos-resistant homokaryotic transformants from the knock-in procedure described for panel A, i.e., the fzo1Q368R:Bar and fzo1WT:Bar transformants, possess a mutant (A1103G) and a wild-type copy of fzo1, respectively. The mus-52 gene in these strains has been disrupted with a hygromycin resistance cassette, which leads to efficient gene targeting. Mutagen sensitivities were tested by spot test analysis as mentioned in the legend to Fig. 1. (D) Complementation experiments. Conidial suspensions of the uvs-5, fzo1Q368R:Bar, and mus-10 strains were spotted onto solid medium as described in the legend to Fig. 1. Suspensions containing mixtures of conidia from the uvs-5 and mus-10 strains (uvs-5 + mus-10), the fzo1Q368R:Bar and mus-10 strains (fzo1Q368R:Bar + mus-10), or the uvs-5 and fzo1Q368R:Bar strains (uvs-5 + fzo1Q368R:Bar) were also spotted onto plates. For these experiments, suspensions containing a total of 104, 103, or 102 conidia (from left to right) were used. (E) Mitochondrial morphologies of hyphae derived from the first subcultures of homokaryotic conidia from the fzo1Q368R:Bar and fzo1WT:Bar strains. The procedure is outlined in the legend to Fig. 2.
Fig 6
Fig 6
Coimmunoprecipitation assays of FLAG-tagged FZO1 and HA-tagged MUS-10. Immunoprecipitation assays were performed in the presence (+) or absence (−) of whole-cell extracts isolated from strains expressing FLAG-tagged FZO1 (FZO1-FLAG) or HA-tagged MUS-10 (MUS-10-HA). When tagged proteins were absent (−), whole-cell extracts from the parental strain (his-3) were used instead. Immunoprecipitation was carried out using anti-FLAG antibodies. Input and immunoprecipitated (IP) samples were separated by SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies.
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