The exception proves the rule? Dual targeting of nuclear‐encoded proteins into endosymbiotic organelles

Abstract

Summary Plant cells harbor two types of endosymbiotic organelle: mitochondria and chloroplasts. As a consequence of endosymbiotic gene transfer, the majority of their proteins are encoded in the nucleus and post‐translationally ‘re’‐imported into the respective target organelle. The corresponding transport signals are usually selective for a single organelle, but several proteins are transported into both the mitochondria and chloroplasts. To estimate the number of proteins with such dual targeting properties in Arabidopsis, we classified the proteins encoded by nuclear genes of endosymbiotic origin according to the respective targeting specificity of their N‐terminal transport signals as predicted by the TargetP software package. Selected examples of the resulting protein classes were subsequently analyzed by transient transformation assays as well as by in organello protein transport experiments. It was found that most proteins with high prediction values for both organelles show dual targeting with both experimental approaches. Unexpectedly, however, dual targeting was even found among those proteins that are predicted to be localized solely in one of the two endosymbiotic organelles. In total, among the 16 candidate proteins analyzed, we identified 10 proteins with dual targeting properties. This unexpectedly high proportion suggests that such transport properties are much more abundant than anticipated.

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

“…in a recent study we showed with a combination of in silico, in organello, and in vivo approaches that the frequency of such proteins is apparently much higher than usually anticipated. 1 here we demonstrate with in organello competition experiments that the import of 2 of these dually targeted proteins (GrpE and EF-tu) takes place by the same import pathways that are used by organelle proteins with "typical" monospecific targeting properties.…”

“…However, the actual number of such dually targeted proteins is probably in the range of several hundred as deduced from both prediction of proteins with dual targeting properties from Physcomitrella patens and a combination of in silico, in organello, and in vivo approaches analyzing the targeting behavior of selected proteins from Arabidopsis thaliana. 1,7 Here, we have analyzed the transport pathways of 2 proteins from Arabidopsis thaliana, notably GrpE (At5g55200) and the elongation factor EF-Tu (At4g02930), that were both recently shown to posses dual targeting properties in transient transformations assays as well as in in organello experiments. 1 Our aim was to investigate whether these proteins are imported along the general import pathways into the 2 endosymbiotic organelles or whether they use transport routes that are different from those of the monospecific organelle proteins.…”

“…1,7 Here, we have analyzed the transport pathways of 2 proteins from Arabidopsis thaliana, notably GrpE (At5g55200) and the elongation factor EF-Tu (At4g02930), that were both recently shown to posses dual targeting properties in transient transformations assays as well as in in organello experiments. 1 Our aim was to investigate whether these proteins are imported along the general import pathways into the 2 endosymbiotic organelles or whether they use transport routes that are different from those of the monospecific organelle proteins. To address this question in organello transport experiments were performed in the presence of increasing amounts of competitor proteins.…”

“…the asterisk marks a degradation product of approximately 38 kDa, which corresponds to a thermolysin-resistant domain of the EF-tu translation product (see ref. 1). the size of molecular marker proteins is given in kDa.…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…These proteins harbor an ambiguous targeting signal (Small et al, 1998;Silva-Filho, 2003;Yogev and Pines, 2011) that induces the concomitant transport to alternative destinations by overlapping targeting signals ( Figure 4C). Such targeting signals have been predominantly found in plant proteins bilocalized to mitochondria and chloroplasts (Carrie and Small, 2013;Baudisch et al, 2014) and use the traditional import pathways into these organelles (Langner et al, 2014). The amino acid composition of N-terminal sequences encoding ambiguous targeting signals show properties of both targeting signals, which emphasizes the intermediate state of such peptides (Pujol et al, 2007).…”