Unique Translation Initiation at the Second AUG Codon Determines Mitochondrial Localization of the Phage-Type RNA Polymerases in the Moss Physcomitrella patens  

Abstract

The nuclear genome of the moss Physcomitrella patens contains two genes encoding phage-type RNA polymerases (PpRPOT1 and PpRPOT2). Each of the PpRPOT1 and PpRPOT2 transcripts possesses two in-frame AUG codons at the 5# terminus that could act as a translational initiation site. Observation of transient and stable Physcomitrella transformants expressing the 5# terminus of each PpRPOT cDNA fused with the green fluorescent protein gene suggested that both PpRPOT1 and PpRPOT2 are not translated from the first (upstream) AUG codon in the natural context but translated from the second (downstream) one, and that these enzymes are targeted only to mitochondria, although they are potentially targeted to plastids when translation is forced to start from the first AUG codon. The influence of the 5#-upstream sequence on the translation efficiency of the two AUG codons in PpRPOT1 and PpRPOT2 was quantitatively assessed using a b-glucuronidase reporter. The results further supported that the second AUG codon is the sole translation initiation site in Physcomitrella cells. An Arabidopsis (Arabidopsis thaliana) RPOT homolog AtRpoT;2 that possesses two initiation AUG codons in its transcripts, as do the RPOTs of P. patens, has been regarded as a dually targeted protein. When the localization of AtRpoT;2 was tested using green fluorescent protein in a similar way, AtRpoT;2 was also observed only in mitochondria in many Arabidopsis tissues. These results suggest that, despite the presence of two in-frame AUGs at the 5# termini of RPOTs in Physcomitrella and Arabidopsis, the second AUG is specifically recognized as the initiation site in these organisms, resulting in expression of a protein that is targeted to mitochondria. This finding may change the current framework of thinking about the transcription machinery of plastids in land plants.

Plant organellar protein targeting: a traffic plan still under construction

“…When one research group included in their studies the coding sequence from the upstream MET, but omitted the untranslated 5′ leader sequence, protein targeting was dual [28]. When a second group tested the same locus by incorporating the 5′ untranslated leader into their reporter gene constructions, initiation occurred at the second MET and targeting was mitochondrial [29,30]. These latter results indicate the importance of gene context in selection of the initiator codon and imply that the protein localizes exclusively to mitochondria.…”

Phage-Type RNA Polymerase RPOTmp Transcribes the rrn Operon from the PC Promoter at Early Developmental Stages in Arabidopsis

“…Several experiments to detect RPOTmp in purified chloroplasts by western-blot analysis were negative (data not shown). The presence of only a very low quantity of RPOTmp in chloroplasts of Arabidopsis might be the reason for the controversial results that have been obtained using GFP fusion constructs to analyze the localization of the enzyme (Hedtke et al, 2000;Kabeya and Sato, 2005).…”

Dual Targeting of Arabidopsis HOLOCARBOXYLASE SYNTHETASE1: A Small Upstream Open Reading Frame Regulates Translation Initiation and Protein Targeting

“…This may reflect a lack of sensitivity in the GFP assay; in case of an uneven dual distribution of a protein, its most abundant localization can impede the detection of the minor one (Duchene et al, 2005). It is also possible that the GFP promoter fusion missed essential cis-elements that aid its targeting to mitochondria but are not necessary for plastidic targeting (Christensen et al, 2005;Kabeya and Sato, 2005). The HCS2 gene does not seem to bear any fundamental function in carboxylase biotinylation in plants, reporting on HCS1 gene, which is essential for plant viability, the whole responsibility of biotin-dependent carboxylase biotinylation within the cell.…”