Abstract
PpRPOT1 and PpRPOT2 transcripts possess two in-frame AUG codons at the N-terminus. Targeting experiments using PpRPOT-GFP fusion proteins suggested that both PpRPOTs were targeted to mitochondria when they were translated from the second methionine, whereas they were targeted to plastids when they were forced to translate from the first one. When the translation efficiency of the two AUG codons in PpRPOT1 and PpRPOT2 was evaluated using GUS reporter, the second AUG codon is used as the sole translation initiation site. Additionally, we showed that the PpRPOT1 and PpRPOT2 are immunologically detected in the mitochondrial fraction but not in the plastids fraction.
We tried to disrupt the PpRPOT1 and the PpRPOT2 genes by homologous recombination, but this was not successful. Therefore, we introduced the PpRPOT1 and PpRPOT2 cDNAs into the Physcomitrella nuclear genome under the control of the strong constitutive E7133 promoter. We currently are analyzing these transformants.
