Dual Targeting of a Mitochondrial Protein: The Case Study of Cytochrome c1

Abstract

As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts is encoded in the nucleus and synthesized in the cytosol as precursor molecules carrying N-terminal transit peptides for the transport into the respective target organelle. In most instances, transport takes place into either mitochondria or chloroplasts, although a few examples of dual targeting into both organelles have been described. Here, we show by a combination of three different experimental strategies that also cytochrome c(1) of potato, a component of the respiratory electron transport chain, is imported not only into mitochondria, but also into plastids. In organello import experiments with isolated mitochondria and chloroplasts, which were analyzed in both single and mixed organelle assays, demonstrate that the processing products accumulating after import within the two endosymbiotic organelles are different in size. Dual targeting of cytochrome c(1) is observed also in vivo, after biolistic transformation of leaf epidermal cells with suitable reporter constructions. Finally, Western analyses employing cytochrome c(1)-specific antiserum provide evidence that the protein accumulates in significant amounts in mitochondria and chloroplasts of both pea and spinach. The possible consequences of our findings on the relevance of the dual targeting phenomenon are discussed.

Organelle import of proteins with dual targeting properties into mitochondria and chloroplasts takes place by the general import pathways

“…These experiments fit well to earlier observations [10][11][12] and reconfirm the conclusion that proteins with dual targeting properties do not use distinct import pathways that were specifically developed for this purpose. This observation can be taken as further hint for the hypothesis that dual targeting is an evolutionary remnant in the development of transit peptides of both endosymbiotic organelles.…”

“…2). Pre-Cytc1 was shown to possess dual targeting properties 10 and, consequently, both monospecific control proteins, mtRieske and FNR, show reduced import into the respective target organelle in the presence of increasing amounts of pre-Cytc1 (Fig. 2C, D).…”

Alternative Oxidases (AOX1a and AOX2) Can Functionally Substitute for Plastid Terminal Oxidase in Arabidopsis Chloroplasts

“…Our proposal that mitochondria and chloroplasts require AOX-like quinol terminal oxidases is consistent with this idea. On the other hand, it is possible that a certain amount of mistargeting might be tolerated by cells as a by-product of the evolutionary development of transit peptides, as suggested in the case of cytochrome c1 of the respiratory chain, which appears to be targeted to chloroplasts as well as mitochondria (Rödiger et al, 2011). The chloroplast targeting of AOX2 and cytochrome c1 suggests that many proteins could have this capacity.…”

“…The chloroplast targeting of AOX2 and cytochrome c1 suggests that many proteins could have this capacity. One evolutionary advantage of this capacity is that it provides a mechanism for neofunctionalization of the organelle, with control exerted by the nucleus (Rödiger et al, 2011). This might have been especially important during the early stages of endosymbiosis.…”

Determining targeting specificity of nuclear-encoded organelle proteins with the self-assembling split-fluorescent protein toolkit

“…For mitochondrial localization of GFP1-10, the N-terminal 100 amino acid residues comprising the presequence of the mitochondrial Rieske Fe/S protein of potato (mtRi1-100; Emmermann et al, 1994) were used. Both transport signals had previously been characterized for their targeting specificity to a single organelle with in vivo and in vitro approaches (Rödiger et al, 2011). For the initial experiments evaluating the suitability of the sasplit-GFP system for our purposes, each transport signal was likewise combined with the GFP11x7 tag (small fragment of sasplit-GFP system).…”