Characterisation of components and mechanisms involved in redox-regulation of protein import into chloroplasts

The vast majority of chloroplast proteins is encoded in the nucleus
and thus has to be posttranslationally imported into the organelle,
a process that is facilitated by two multimeric protein
machineries, the Toc and Tic complexes (translocon at the
outer/inner envelope of chloroplasts). Regulation of protein
import, e.g. by redox signals, is a crucial step to adapt the
protein content to the biochemical requirements of the organelle.
In particular, one subunit of the Tic complex, Tic62, has been
proposed as a redox sensor, whose possible function is to regulate
protein import by sensing and reacting to the redox state of the
organelle. To elucidate a potential redox regulation of protein
import, structural features, redox-dependent properties and the
evolutional origin of Tic62 were investigated. The results show
that Tic62 consists of two very different modules: the N-terminal
part was found to be mainly -helical and possesses dehydrogenase
activity in vitro. It is furthermore an evolutionary ancient
domain, as it is highly conserved in all photosynthetic organisms
from flowering plants to cyanobacteria and even green sulfur
bacteria. In contrast to this, the C-terminus is largely disordered
and interacts specifically with ferredoxin-NADP+ oxidoreductase
(FNR), a key enzyme in photosynthetic electron transfer reactions.
Moreover, this domain was found to exist only in flowering plants,
and thus the full-length Tic62 protein seems to be one of the
evolutionary youngest Tic components. The results of this study
make also clear that Tic62 is a target of redox regulation itself,
as its localization and interaction properties depend on the
metabolic redox state: oxidized conditions lead to fast membrane
binding and interaction with the Tic complex, whereas reduced
conditions cause solubilization of Tic62 into the stroma and
increased interaction with FNR. This novel shuttling behaviour
indicates a dynamic composition of the Tic complex. The NADP+/NADPH
ratio was furthermore found to be able to influence the import
efficiency of many precursor proteins. Interestingly, the import of
not all preproteins depends on the stromal redox state. Hence it
was proposed that not a single stable Tic translocon exists, but
several Tic subcomplexes with different subunit compositions, which
might mediate the import of different precursor groups in a
redox-dependent or -independent fashion. Another redox signal that
was analyzed in regard to an impact on protein import is the
reversible reduction of disulfide bridges, which was found to
affect the channel and receptor proteins of the Toc complex. The
import of all proteins that use the Toc translocon for entering the
chloroplast was shown to be influenced by disulfide bridge
formation. Thus it can be concluded that a variety of redox
signals, acting both on the Toc and Tic complexes, are able to
influence chloroplast protein import.