Comparative analysis of putative orthologues of mitochondrial import motor subunit: Pam18 and Pam16 in plants

Abstract

Pam18/Tim14 and Pam16/Tim16, highly conserved proteins among eukaryotes, are two essential subunits of protein import motors localized in the inner mitochondrial membrane. The heterodimer formed by Pam18 and Pam16 via their J-type domains serves a regulatory function in protein translocation. Here, we report that thirty-one Pam18 and twenty-six Pam16 putative orthologues in twelve plant species were identified and analyzed through bioinformatics strategy. Results data revealed that Pam18 and Pam16 were also highly conserved among plants including their J-type domains within the hydrophilic region. Key amino acid residues and an HPD motif of Pam18 were identical among the orthologues except OsPam18L5. N-myristoylation sites of Pam18 and casein kinase II phosphorylation sites of Pam 16 were more abundant, which might be important functional sites. Some Pam18 and Pam16 proteins contained a transmembrane region at the N-terminal region. Sub-cellular prediction results indicated that many orthologues localized at mitochondria. Gene expression analyses revealed that Pam18 and Pam16 in Arabidopsis might play roles in senescence and abiotic stress responses. Our detailed study provides a better understanding of Pam18 and Pam16 in plant kingdom.

Figure 1. Phylogenetic tree analysis of putative Pam18 (A) and Pam16 (B) orthologues from twelve plant species.

Both Pam18 (A) and Pam16 (B) phylogenetic trees were generated by the neighbor-joining method and bootstrap values (1000 replications) are indicated at each branch. The unit bar on the tree represents the measure of phylogenetic distance.

Figure 2. Amino acid sequence alignment of Pam18 (A) and Pam16 (B) in Saccharomyces cerevisiae and twelve plant species.

Only the conserved regions are shown. Identical amino acid residues were shaded black and similar amino acid residues were shaded grey. J-domain of Pam18 (A) and J-like domain of Pam16 (B) are indicated in the red rectangle. An HPD motif is shown and two important phenylalanine residues are indicated by asterisk (A). ScPam18 and ScPam16 protein sequence were used for comparison.

Figure 3. Amino acid composition of Pam18 and Pam16 in twelve plant species (A) and Saccharomyces cerevisiae (B).

Thirty-one amino acid sequences of Pam18 were combined together to evaluate average amino acid composition using Bioedit 7.0 software. The same strategy was adopted in Pam16 amino acid evaluation (Twenty-six sequences in total). Histograms show the Molar percent of each residue of the combined sequence. Amino acid composition of ScPam18 and ScPam16 is shown for comparison.

Figure 4. Mean Hydrophobicity profiles of Pam18 and Pam16.

Thirty-one aligned amino acid sequences (Pam18) including ScPam18 and twenty-six aligned amino acid sequences (Pam16) including ScPam16 were used to evaluate hydrophobicity using Bioedit 7.0 software. ScPam18 and ScPam16 are indicated by a red line.

Figure 5. Expression pattern of AtPam18 and AtPam16 at developmental stages.

Relative expressions of AtPam and At3G04800 were determined by real-time qRT-PCR. Expression levels were normalized by Actin-2. Error bars represent means of three replicates ± SD. Similar results were obtained from three independent replicates and one representative result is shown (A). Expression profiles as heat map (B) in AtPam and putative genes coding AtTim44, AtTim23, AtTim17 and AtTim50 were generated by Genevestigator.

Figure 6. Expression pattern of AtPam18 and AtPam16 under cold, heat, salt and drought stresses.

Relative expressions of AtPam and At3G04800 were determined by real-time qRT-PCR. Expression levels were normalized by Actin-2. Error bars represent means of three replicates ± SD. Asterisks indicate significant differences of cold, heat, salt and drought treatments compared with control based on Student’s t test, P<0.05 (*), P<0.01 (**). One of three independent experimental replicates with similar results is shown (A). Fold-change expression levels (B) in AtPam and putative genes coding AtTim44, AtTim23, AtTim17 and AtTim50 were from Genevestigator.From these results, it is speculated that AtPam genes might play key roles in plant senescence and heat stress responses.