Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import

A deletion and mutagenesis study was performed on the mitochondrial presequ ence of the beta -subunit of the F-1-ATP synthase from Nicotiana plumbagini folia linked to the green fluorescent protein (GFP). The various constructs were tested in vivo by transient expression in tobacco protoplasts. GFP di stribution in transformed cells was analysed in situ by confocal microscopy , and in vitro in subcellular fractions by Western blotting. Despite its be ing highly conserved in different species, deletion of the C-terminal regio n (residues 48-54) of the presequence did not affect mitochondrial import. Deletion of the conserved residues 40-47 and the less conserved intermediat e region (residues 18-39) resulted in 60% reduction in GFP import, whereas mutation of conserved residues within these regions had little effect. Furt her shortening of the presequence progressively reduced import, with the co nstruct retaining the predicted N-terminal amphiphilic alpha -helix (residu es 1-12) being unable to mediate mitochondrial import. However, point mutat ion showed that this last region plays an important role through its basic residues and amphiphilicity, but also through its hydrophobic residues. Rep lacing Arg4 and Arg5 by alanine residues and shifting the Arg5 and Leu6 (in order to disturb amphiphilicity) resulted in reduction of the presequence import efficiency. The most dramatic effects were seen with single or doubl e mutations of the four Leu residues (positions 5, 6, 10 and 11), which res ulted in marked reduction or abolition of GFP import, respectively. We conc lude that the N-terminal helical structure of the presequence is necessary but not sufficient for efficient mitochondrial import, and that its hydroph obic residues play an essential role in in vivo mitochondrial targeting.