Characterization of the protein import pathway in pea chloroplast

In order to sustain their structure and metabolism, chloroplasts
and other plastid types must import the majority of their proteins
from the cytosol across the envelope membrane. Translocation of
these precursor proteins across the double envelope membrane is
achieved by two multimeric complexes - the so-called TOC and TIC
complexes (Translocon at the Outer envelope of Chloroplast and
Translocon at the Inner envelope of Chloroplast, respectively).
N-terminal transit peptides essential for import of the precursor
proteins are cleaved after their entry into the stroma. It was thus
far believed that all of the different cytosolic precursor proteins
would enter the chloroplast through the same, jointly acting
TOC/TIC machineries. Recent evidence, however, suggests that
multiple, regulated import pathways exist in plastids that involve
different import machineries. Different combinations of TOC and TIC
proteins were shown to establish different import sites in
Arabidopsis thaliana with specificity for either photosynthetic
proteins (the general import pathway) or non-photosynthetic
„housekeeping“ proteins. Moreover, numerous non-canonical import
pathways such as the import of Tic32 and AtQORH mediated by the yet
unknown novel import pathway and the import via the secretory
pathway were shown to exist. Proteomics studies have revealed the
presence of a large number of plastid proteins lacking predictable
N-terminal transit sequences for import. The import mechanism for
the majority of these proteins has not been determined yet.
Examples of the transit sequenceless precursor proteins are the
chloroplast envelope quinone oxidoreductase homologue, AtQORH and
the chloroplast inner envelope protein 32, Tic32. Both proteins are
imported into the inner plastid envelope membrane by a
non-canonical pathway (Toc159- and Toc75-independent) and without
any proteolytic cleavage. In the present study not only the import
characteristic of nine tentative ‘non-canonical’ chloroplast
precursor proteins but also the new interactions between these
precursor proteins and the proteins at the organellar surfaces were
analyzed. Moreover, a non-canonical precursor protein without the
classical transit peptide, the iron superoxide dismutase (FSD1)
could be identified. Biochemical crosslinking experiments revealed
that FSD1 interacts with new members of the Toc159 family in pea,
namely PsToc132 and PsToc120. Using deletion mutants as well as a
peptide scanning approach, regions of the precursor protein, which
are involved in receptor binding could be defined. These are
distributed across the entire sequence; surprisingly only the
extreme N-terminus as well as a C-proximal domain turned out to be
essential for targeting and import. En route into the plastid FSD1
engages components of the general import pathway, implying that in
spite of the ‘non-canonical’ targeting information and recognition
by a specific receptor, this precursor protein follows a similar
way across the envelope as the majority of plastid precursor
proteins.