About 99% of mitochondrial proteins are encoded for by nuclear genes, synthesised in the cytosol and imported into the organelle. Specific translocases have evolved in the outer and inner mitochondrial membranes to sort nuclear encoded proteins to one of the four mitochondrial compartments; the outer membrane, the intermembrane space, the inner membrane and the matrix. Tom40, the core component of the TOM (Translocase of the Outer Membrane) complex forms the central channel for precursor translocation across the mitochondrial outer membrane and is essential for cell viability. Given the indispensable role of Tom40 in protein import into mitochondria, mutations in the mammalian Tom40 gene (TOMM40) are likely to have pathological consequences. Screening of mutant mice generated by Ethylnitrosourea (ENU) mutagenesis revealed a TOMM40 mutant mouse strain. None of the homozygous TOMM40 muant mice has lived more than 7 weeks of age, the post mortem analysis revealing pulmonary vascular congestion. Catheterisation of the right ventricle showed elevated pressure, consistent with pulmonary hypertension. Genome sequencing of the mouse unveiled a mutation in the TOMM40 gene resulting in a substitution of an alanine for a valine. In vitro studies showed that the single mutation in Tom40 affects the stability of the TOM complex. In vitro import assays revealed an assembly defect in an inner membrane carrier protein, Adenine Nucleotide Translocase1 (ANT1) specific to heart tissues. The broader aim of this work is to decipher how the alanine to valine substitution in Tom40 contributes to the severe phenotype observed in mice.