Protein import into the inner envelope membrane of chloroplasts

Most of the proteins localized in the chloroplast inner envelope
membrane are synthesized on cytosolic ribosomes with a cleavable
N-terminal chloroplast transit peptide. Most of them reach their
final localization via the so called general import pathway
consisting of the Toc complex at the chloroplast outer envelope
membrane and the Tic complex at the chloroplast inner envelope
membrane. Recent studies characterized precursor proteins which are
targeted into the chloroplast inner envelope membrane by two
different import pathways. The first route, called “conservative
sorting”, was described for Tic40 and Tic110, which prior to inner
envelope membrane insertion reach the stroma. The second route,
called “stop-transfer” was proposed for ARC6, which is arrested at
the level of the inner envelope membrane and probably laterally
inserted into the lipid bilayer. Taking into consideration both
import mechanisms we characterized import pathways of nine
chloroplast inner envelope membrane proteins containing cleavable
transit peptides and a different number of hydrophobic -helices.
On the basis of the results observed in the stromal processing
assays as well as results obtained in the pulse-chase experiments,
within investigated precursor proteins two classes could be
distinguished. The first class consisted of precursors processed
once to their mature forms, i.e. containing a “single” transit
peptide, whereas the second class consisted of precursors processed
twice to the intermediate and the mature form, i.e. containing a
bipartite transit peptide. In the processing of almost all
precursor proteins stromal processing peptidase (SPP) was involved.
Most probably at least one protein containing a bipartite transit
peptide was also processed by another peptidase present not in the
stromal compartment. We showed that despite of the differences in
the number of hydrophobic transmembrane segments and different
types of transit peptides, all investigated proteins had similar
import properties. Their import was dependent on outer envelope
membrane receptors and mediated by the general import pathway at
least in the initial import phase. All investigated proteins
required energy for import. 200 M ATP was sufficient for proteins
used in this study to achieve the maximal import rate.
Interestingly, neither intermediates nor mature proteins were
extractable from the membrane by urea treatment and all proteins
seemed not to possess a soluble import intermediate. Therefore we
claim that all investigated precursor proteins were imported via
the “stop-transfer” pathway. Moreover, most probably at least some
components of the Tic complex were involved in the transport of
precursor proteins at the level of the inner envelope membrane and
the process was Ca2+/calmodulin regulated.