Simple prerequisite of presequence for mitochondrial protein import in the unicellular red alga Cyanidioschyzon merolae

Mitochondrial biogenesis relies on hundreds of proteins which are derived from genes encoded in the nucleus. According to characteristic properties of N-terminal targeting peptides (TP) and multi-step authentication by the protein translocase called the TOM complex, nascent polypeptides satisfying the requirements are imported into mitochondria. However, it has not been investigated whether the eukaryotic cell with a simple proteome and a single mitochondrion in a cell has a similar or simpler complexity of presequence requirements for mitochondrial protein import as other eukaryotes with multiple mitochondria. Based on the amino acid compositions of putative mitochondrial TP sequences in the unicellular red alga Cyanidioschyzon merolae, we designed the synthetic TP (synTP) and confirmed that synTP-fused mVenus were translocated into the mitochondrion in vivo. Through a series of experimental evaluations using modified synTPs, we showed that functional TP must have some basic residues, at least one, and compose the specific amino acid composition, but the physicochemical properties of net charge, hydrophobicity and hydrophobic moment are not strictly determined in C. merolae. Combined with the simple composition of the TOM complex in C. merolae, our results suggest that a regional positive charge in TP would be recognized and verified solely by TOM22 as a single-step authentication for mitochondrial protein import in C. merolae. The simple authentication mechanism indicates that the C. merolae cell, with its simple cell structure and genome, would not need to increase the cryptographic complexity of the lock-and-key for mitochondrial protein import.