Demonstration that the Neurospora crassa mutation un-4 is a single nucleotide change in the tim16 gene encoding a subunit of the mitochondrial inner membrane translocase

Aric Wiest, Michael Plamann, and Kevin McCluskey
Fungal Genetics Stock Center, School of Biological Sciences, University of Missouri-Kansas City

Fungal Genetics Reports 55:37-39
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The Neurospora crassa temperature sensitive mutation known as un-4 has been shown by a map-based complementation approach to be a single nucleotide change in the open reading frame of the mitochondrial inner membrane translocase subunit tim16 (NCU05515). 

Many mutations in Neurospora crassa are only known by a morphological or other visible phenotype. For many of these, the actual open reading frame responsible  remains unknown. Among these are several temperature-sensitive lethal mutations known as unknown (Inoue and Ishikawa, 1970; Ishikawa and Perkins, 1983). As part of our continuing effort to define the gene defect associated with these otherwise anonymous temperature sensitive mutations, we have identified un-4 as a missense mutation in the tim16 (translocase of the inner mitochondrial membrane) gene. We used a complementation approach to identify an open reading frame that conferred the ability to grow at 37C on the un-4 strain FGSC 2172.  Since Schmidhauser et al.  (1999) reported that un-4 was on a cosmid with lys-5, we attempted to complement the un-4 lesion using  cosmid G13:G8 from the Orbach Sachs pMOcosX library (Orbach and Sachs, 1991; Vollmer and Yanofsky, 1986). While this cosmid was reported to complement un-4 (Schmidhauser et al., 1999), it did not do so in our hands (Table 1). Indeed, in assembly 7 of the Neurospora genome (Galagan et al., 2003), this cosmid maps to linkage group IV on contig 43, while un-4 and lys-5 are on linkage group VI. Based on the location of lys-5 (NCU05526) on contig 22, we chose several cosmids from contig 22 and found that two did restore the ability to grow at 37°C to the un-4 strain FGSC 2172 (Table 1). Three genes were identified as possible candidates based on the overlapping regions of complementing cosmids. These three candidate genes were amplified with PCR and tested for their ability to complement the un-4 mutation (Table 2). This approach allowed the identification of NCU05515.3 as the likely open reading frame that is mutated in the un-4 strain.
 

Table 1.  Identification of cosmids that complement un-4. All cosmids tested carry the hygromycin resistance cassette.
^a (Kelkar et al., 2001)

 DNA sequence obtained directly from PCR amplified genomic DNA from strain 2172 showed a single C to T transition at position 293 of the coding sequence of NCU05515.3. This results in a serine to phenylalanine change in the polypeptide at position 98.  The serine at position 98 is conserved among most fungi but in some higher eukaryotes, this position is occupied by a threonine (Figure 1). This region is part of a J-like domain and is thought to interact with Tim14 via hydrogen bonds (Mokranjac et al., 2006).

Table 2.  Identification of PCR products that complement un-4. Data from two transformation experiments are shown.

Figure 1.  Alignment of amino acid sequence from the J-like region of Tim16 among fungi and select eukaryotes. Position 98 is indicated by the carat (^) underneath. M. hisrutus is Maconellicoccus hirsutus, also known as the hibiscus mealybug. Caenorhabditis elegans and C. briggsae are both nematodes. Ostreococcus tauri is a unicellular green alga.

The demonstration that un-4 defines the tim16 gene adds value to strains carrying this mutation. The ability to study the interactions of subunits of the mitochondrial protein import motor will be enhanced by the ability to use a temperature-sensitive mutation to control the action of the motor.

 
Acknowledgement

The FGSC is supported by award number 0235887 from the National Science Foundation and receives additional funds from award 5P01GM068087-04 from the National Institutes of Health.

References

Galagan, James E., et al.  2003. The genome sequence of the filamentous fungus Neurospora crassa.  Nature 422: 859 - 868.

Inoue, H., and T. Ishikawa. 1970. Macromolecule synthesis and germination of conidia in temperature-sensitive mutants of Neurospora crassa. Jpn. J. Genet. 45: 357-369.

Ishikawa, T. and D. D. Perkins. 1983. Additional irreparable temperature-sensitive mutants. Neurospora Newl. 30:9

Kelkar, H.S., J. Griffith, M.E. Case, S.F. Covert, R.D. Hall, C.H. Keith, J.S. Oliver, M.J. Orbach, M.S. Sachs, J.R. Wagner, M.J. Weise, J.K. Wunderlich, and J. Arnold. 2001. The Neurospora crassa genome: cosmid libraries sorted by chromosome. Genetics 157: 979-990.

Mokranjac, D., G. Bourenkov, K. Hell, W. Neupert and M. Groll. 2006. Structure and function of Tim14 and Tim16, the J and J-like components of the mitochondrial protein import motor. The Embo Journal 25:4675-4685

Orbach, M.J. and M.S. Sachs. 1991. The Orbach/Sachs cosmid library of N. crassa DNA sequences (pMOcosX). Fungal Genet. Newsl. 38: 97.

Schmidhauser, T. J., D. Chen and Y. Wan. 1999. Identification of a cosmid clone containing the Neurospora crassa lys-5 and un-4 genes, isolation of a partial lys-5 cDNA and associated chromosome walking. Fungal Genet. Newsl. 46:23-24

Vollmer, S. J. and C. Yanofsky. 1986. Efficient cloning of genes of Neurospora crassa. Proc. Natl. Acad. Sci. USA. 83: 4869-4873.

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