Abstract
Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA) from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement), we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene.
Highlights
Mitochondria are key organelles of eukaryotic cells involved in energy production, metabolism, and signaling
Proteins of the LEA_3 Family Are Expected to Be Mitochondrial In Arabidopsis, the LEA_3 family comprises four proteins, with three of them being exclusively mitochondrial, while the fourth (LEA2) is cytosolic [25]. This suggests that LEA2, which is paralogous to LEA38, could have lost its mitochondrial localization during evolution
The LEA_3 family is specific to higher plants, and both experimental data in Arabidopsis [25] and targeting predictions for 390 proteins of the family strongly suggest that mitochondrial localization is a characterizing feature of this family
Summary
Mitochondria are key organelles of eukaryotic cells involved in energy production, metabolism, and signaling. Precursors for inner membrane proteins do not exhibit an MTS but internal targeting signals [3,4]. Protein import in plastids proceeds in a similar way as in mitochondria, but with different translocators (for review, [10]), and it is not surprising that mitochondrial and plastidial pre-sequences share some common features and are not distinguished, mitochondrial MTS generally contain more arginine [11]. MTS share common properties because they carry essential information for proper targeting of several hundred mitochondrial proteins. They are still insufficiently characterized, partly because of their structural diversity, and because of their similarity to plastid transit peptides. MTS contain a high proportion of positively charged, hydroxylated, and hydrophobic residues, but few acidic amino acids [14], and plant pre-sequences are noticeably enriched in serine [15]
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