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MBE Advance Access published online on May 30, 2003

Molecular Biology and Evolution, doi:10.1093/molbev/msg132
Molecular Biology and Evolution © Society for Molecular Biology and Evolution 2003; all rights reserved
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Accepted March 24, 2003
© 2003 Society for Molecular Biology and Evolution

Original Articles

Functional Respiratory Chain Analyses in Murid Xenomitochondrial Cybrids Expose Coevolutionary Constraints of Cytochrome b and Nuclear Subunits of Complex III

Matthew McKenzie 1, Maria Chiotis 1, Carl A. Pinkert 2, Ian A. Trounce 3*

1 Genomic Disorders Research Centre, University of Melbourne, Department of Medicine, St. Vincent's Hospital, Victoria 3065 Australia
2 Department of Pathology and Laboratory Medicine, Center for Aging and Developmental Biology, University of Rochester Medical Center, New York
3 Genomic Disorders Research Centre, University of Melbourne, Department of Medicine, St. Vincent's Hospital, Victoria 3065 Australia; Centre for Neuroscience, University of Melbourne, Victoria 3010 Australia

* To whom correspondence should be addressed. E-mail: i.trounce{at}unimelb.edu.au.


   Abstract

The large number of extant Muridae species provides the opportunity of investigating functional limits of nuclear/mitochondrial respiratory chain (RC) subunit interactions by introducing mitochondrial genomes from progressively more divergent species into Mus musculus domesticus mtDNA-less ({rho}0) cells. We created a panel of such xenomitochondrial cybrids, using as mitochondrial donors cells from six murid species with divergence from M. m. domesticus estimated at 2 to 12 million years before present. Species used were Mus spretus, Mus caroli, Mus dunni, Mus pahari, Otomys irroratus and Rattus norvegicus. Parsimony analysis of partial mtDNA sequences showed agreement with previous molecular phylogenies, with the exception that Otomys did not nest within the murinae as suggested by some recent nuclear gene analyses. Cellular production of lactate, a sensitive indicator of decreased respiratory chain ATP production, correlated with divergence. Functional characterization of the chimeric RC complexes in isolated mitochondria using enzymological analyses demonstrated varying decreases in activities of complexes I, III and IV which have subunits encoded in both mitochondrial and nuclear genomes. Complex III showed a striking decline in electron transfer function in the most divergent xenocybrids, being greatly reduced in the Rattus xenocybrid and virtually absent in the Otomys xenocybrid. This suggests that nuclear subunits interacting with cytochrome b face the greatest constraints in the coevolution of murid RC subunits. We sequenced the cytochrome b gene from the species used, to identify potential amino acid substitutions involved in such interactions. The greater sensitivity of complex III to xenocybrid dysfunction may result from the encoding of redox centre apoproteins in both nuclear and mitochondrial genomes, a unique feature of this RC complex.

Key Words: cybrid, xenomitochondrial, cytochrome b, mtDNA, respiratory chain, coevolution, murids


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