Neurospora Mutants Affecting the Respiratory Pathway
(special thanks to Frank Nargang for help in writing these descriptions)Below is a brief description of the function and assembly of the multi-subunit respiratory complexes that require coordinated expression of both mitochondrial and nuclear genes (complexes I, III, IV and V), known mutants of Neurospora crassa that have an effect on the function of one or more complex, and the role the alternative components may play in respiratory function. Mutants affecting the function and assembly of the respiratory pathway are listed in the Table below.
Complex I is a large multisubunit complex that spans the inner mitochondrial membrane and is comprised of seven mitochondrial-encoded gene products (nad 1, 2, 3, 4, 4L, 5, 6) and more than 25 nuclear-encoded products. This complex is well-studied in Neurospora and thought to be very similar to the equivalent complex found in animals. Examination of the Neurospora [E35] stopper mutant revealed that the strain contained two different types of deletion-bearing mtDNAs and that the common region deleted encompassed the nad2 and nad3 genes. This strain was shown to have defects in the assembly of the membrane arm of complex I which includes the mtDNA encoded subunits of the enzyme (Alves and Videira 1998). The mitochondrially encoded NAD5 subunit of the complex has been shown to be myristoylated, though the role of the modification is unknown (Plesofsky et al. 2000). Mutations in the nuclear nuo genes have been developed by targeted gene mutagenesis and shown to affect the assembly and activity of complex I (Videira 1998; Perkins et al. 2001). These mutants are all viable in the vegetative state, though complex I activity is required for successful completion of the sexual cycle (Duarte and Videira 2000).
In addition to the large multi-subunit complex I, Neurospora mitochondria also contain alternative dehydrogenases that are encoded by single nuclear genes (Melo et al. 2001). These gene products appear to be located on either the matrix (internal) or intermembrane space (external) side of the inner membrane. Having both internal and external alternative dehydrogenases could allow electrons to enter the respiratory chain from either side of the membrane and it is thought that this may help reduce the production of reactive oxygen species generated by the standard respiratory pathway (Joseph-Horne at al. 2001).
Complex III contains nine subunits, of which only the gene for apocytochrome b is encoded in the mitochondrion. There are no mitochondrial mutations directly affecting the cob locus in Neurospora. The nuclear cyb mutants lack cytochrome b, but it is unknown if these mutations affect a complex III subunit or factors required for the synthesis or assembly of cytochrome b.
Cytochrome c exists in the intermembrane space and passes electrons between complexes III and IV. The protein is encoded by the nuclear cyc-1 gene in Neurospora (Bottorff et al. 1994). The cyt-2 nuclear gene encodes cytochrome c heme lyase, which attaches the heme group to the cytochrome c polypeptide and the primary lesion in the cyt-2-1 mutant is known to affect cytochrome c heme lyase activity (Drygas et al., 1989).
Complex IV is the terminal oxidase of the standard respiratory pathway. The Neurospora complex contains seven to eight polypeptides (Werner 1977) of which three (COX1, 2, 3) are mitochondrially encoded. In the [mi-3] mutant, a mutation resulting in a single amino acid change in the COX1 protein is thought to produce the mi-3 phenotype. Like the NAD5 subunit of complex I, the COX1 polypeptide of complex IV is also covalently linked to a myristic acid moiety in Neurospora (Vassilev et al. 1995). It was suggested that one role of the modification might be to aid in the assembly of complex IV subunits with the COX1 product. The [exn-5] mutant is deficient in the COX2 subunit due to a single amino acid change in the coding sequence (Lemire et al. 1991). There are no mutants of the cox3 gene yet identified in Neurospora. The cya-4 nuclear gene specifies subunit 5 of the complex (Sachs et al. 1989).
Complex V is the mitochondrial ATP synthase. There are no point mutations known to affect the atp6 or atp8 genes, which encode two of the ATP synthase subunits in Neurospora. However, the temperature-sensitive mutant [C93] is selectively deficient in the protein encoded by atp6 when grown at its non-permissive temperature. Also, growth of [C93] is very sensitive to low concentrations of the ATP synthase inhibitor oligomycin. These observations raise the possibility that the primary defect in the [C93] mutant may be in the function or assembly of the ATP synthase complex (Collins et al. 1981).
One other nuclear gene has been characterized which has an indirect affect on respiratory function as it is required for the maintenance of mtDNA in vegetative cells (Seidel-Rogol et al. 1989; Bertrand et al. 1993). Other nuclear mutants affecting the respiratory pathway have been isolated but are not yet fully characterized (Bertrand et al. 1977; Perkins et al. 2001).
Genetic studies of the interactions between various nuclear and mitochondrial mutants have also revealed complex regulatory circuits that affect the expression or assembly of the respiratory components that are summarized in Griffiths et al. (1995).
Nuclear genes affecting mitochondrial gene expression and/or respiratory pathway in Neurospora.
|
Process |
Gene |
Product, if known[1] |
Mutant phenotype/gene function |
Reference |
|
Transcription |
cyt-5 |
mt RNA polymerase |
SG, def/mt transcription |
Chen et al. 1996 |
|
|
su([mi-1])-1,3,4,5,10,14; f |
|
suppress [poky] mutations, affect transcription |
Collins & Bertrand 1978; Kubelik et al. 1990 |
|
|
|
|
|
|
|
RNA Splicing/ Processing |
cyt-4 |
ribonuclease II |
SG, def/5' and 3' RNA processing |
Turcq et al. 1992 |
|
|
cyt-18 |
mt tyrosyl-tRNA synthetase |
SG, def/aminoacylation , Group I intron splicing |
Akins & Lambowitz 1987 |
|
|
cyt-19 |
DEAD-box protein |
SG, def/Group I intron splicing, processing |
Mohr et al. in press |
|
|
cyt-20 |
nuclear and mt valyl-tRNA synthetase |
SG, def |
Kubelik et al. 1991 |
|
|
|
|
|
|
|
Assembly/ Translation |
cya-4 |
Subunit 5 of complex IV |
SG, cyt aa3 deficient/structural gene |
Sachs et al. 1989 |
|
|
cya-5 |
homology to PET309 |
SG, cyt aa3 deficient/cox1 post-transcriptional expression |
Coffin et al. 1997 |
|
|
cyb-1 |
|
SG, cyt b deficient |
Bertrand et al. 1977 |
|
|
cyb-2 |
|
SG, cyt b deficient |
Bertrand et al. 1977 |
|
|
cyt-21 |
S-mt-rib-sub protein |
|
Kuiper et al. 1988 |
|
|
cyt-22 |
L-mt-rib-sub protein |
|
Kubelik et al. 1989 |
|
|
cyt-U-28 |
|
SG, def, deficient in L-mt-rib-sub |
Videira et al. 1988 |
|
|
KTS,LAE |
|
SG, def, deficient in mt ribosomes |
Nargang et al. 1979 |
|
|
leu-5 |
mt leucyl-tRNA synthetase |
|
Chow et al. 1989 |
|
|
nuo genes |
complex I subunits |
mild growth defects, required for sexual development/structural genes |
Videira 1998 Perkins et al. 2001 |
|
|
|
|
|
|
|
Other |
cyc-1 |
cyt c structural gene |
cyt aa3 and c deficient/cyt c gene |
Bottoroff et al. 1994 |
|
|
cyt-2-1 |
cyt c heme lyase |
cyt aa3 and c deficient/cyt c heme lyase activity |
Drygas et al. 1989 |
|
|
su([mi-3])-1 |
|
suppresses [mi-3], [exn-5] |
Bertrand & Pittenger 1972 |
|
|
|
|
|
|
|
mtDNA stability |
nd |
|
SG, def, cell death with mtDNA degeneration |
Bertrand et al. 1993 |
|
|
sen |
|
SG, def, cell death |
Navaraj et al. 2000 |
A version of this Table will be published in "The Mycota, Vol. II 'Genetics and Biotechnology' second edition." U. Kuck (ed.) Springer-Verlag (2003).
[1] Abbreviations used: cyt, cytochrome; def, deficiency of both cytochromes aa3 and b; SG, slow growth; S-mt-rib-sub, small mitochondrial ribosomal subunit; L-mt-rib-sub, large mitochondrial ribosomal subunit; mt, mitochondrial.
REFERENCES
| Alves PC, Videira A (1998) The membrane domain of complex I is not assembled in the stopper mutant E35 of Neurospora. Biochem Cell Biol 76: 139-143 and c in Neurospora crassa. Curr Genet 26: 329-335. |
| Bertrand H, Nargang FE, Collins RA, Zagozeski CA (1977) Nuclear cytochrome-deficient mutants of Neurospora crassa: isolation, characterization, and genetic mapping. Molec Gen Genet 153: 247-257 |
| Bertrand H, Wu Q, Seidel-Rogol BL, (1993) Hyperactive recombination in the mitochondrial DNA of the natural death nuclear mutant of Neurospora crassa. Mol Cell Biol 13: 6778-6788 |
| Bottorff DA, Parmaksizoglu S, Lemire EG, Coffin JW, Bertrand H, Nargang FE (1994) Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa3 and c in Neurospora crassa. Curr Genet 26: 329-335. |
| Collins RA, Bertrand H, LaPolla RJ, Lambowitz AM (1981) A novel extranuclear mutant of Neurospora with a temperature sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase. Molec Gen Genet 181: 13-19. |
| Drygas ME, Lambowitz AM, Nargang FE (1989) Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase. J Biol Chem 264:17897-17906. |
| Griffiths AJF, Collins RA, Nargang FE (1995) Mitochondrial genetics of Neurospora. In, K¸ck U (ed) The Mycota II Genetics and Biotechnology. Springer-Verlag, Berlin Heidelberg, pp 93-105 |
| Joseph-Horne T, Hollomon DW, Wood PW (2001) Fungal respiration: a fusion of standard and alternative components. Biochim Biophys Acta 1504: 179-195. |
| Lemire EG, Nargang FE (1986) A missense mutation in the oxi-3 gene of the [mi-3] extranuclear mutant of Neurospora crassa. J Biol Chem 261:5610-5615. |
| Lemire EG Percy JA, Correia JM, Crowther BM, Nargang FE (1991) Alteraton of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa. Curr Genet 20:121-127. |
| Melo AM, Duarte M, Moller IM, Prokisch H, Dolan PL, Pinto L, Nelson MA and Videira A (2001). The external calcium-dependent NADPH dehydrogenase from Neurospora crassa mitochondria. J Biol Chem 276: 3947-3951. |
| Perkins DD, Radford A, Sachs MS (2001) In, The Neurospora Compendium. Academic Press, San Diego, London |
| Plesofsky N, Gardner N, Videira A, Brambl R (2000) NADH dehrydrogenase in Neurospora crassa contains myristic acid covalently linked to the ND5 subunit polypeptide. Biochim Biophys Acta 1495: 223-230 |
| Sachs MS, Bertrand H, Metzenberg RL, RajBhandary UL (1989) Cytochrome oxidase subunit V of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V. Mol Cell Biol 9: 566-577. |
| Seidel-Rogol BL, King J, Bertrand H (1989) Unstable mitochondrial DNA in natural-death nuclear mutants of Neurospora crassa. Mol Cell Biol 9:4259-4264. |
| Vassilev AO, Plesofsky-Vig N, Brambl R (1995) Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1. Proc Natl Acad Sci USA 92: 8680-8684 Videira A (1998) Complex I from the fungus Neurospora crassa. Biochim Biophys Acta 1364: 89-100 |
| Werner S (1977). Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa. Eur J Biochem 79(1): 103-110. |
