Stimulation of Transit-peptide
Release and ATP Hydrolysis by a
Co-chaperone during Protein
Import into Chloroplasts
Abstract / Three components of the chloroplast protein translocon, Tic110, Hsp93 and Tic40, have
been shown to be important for protein translocation across the inner envelope membrane into the
stroma. Here we show that transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which
in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing.
The Tic40 C-terminal domain, which is homologous to the C terminus of co-chaperones Sti1p/Hop
and Hip but with no known function, stimulates ATP hydrolysis by Hsp93. Hsp93 dissociates from
Tic40 in the presence of ADP, suggesting that Tic40 functions as an ATPase activation protein for
Hsp93. Our data provide the first model for sequential steps of protein translocation into the chloroplast stroma.
Hsou-Min Li
Institute of Molecular Biology, Academia Sinica
Tic110 is the major Tic component identified. It is thought to be the stroma-side receptor
for transit peptides and the first protein that binds
precursors as they emerge from the inner membrane channel. Hsp93 is proposed to function as
the motor that uses the energy of ATP hydrolysis
to translocate proteins into the stroma. Tic40 has
an N-terminus membrane anchor and a stromalocated hydrophilic domain, which is composed
of a TPR domain and a C terminal-domain
homologous to the C terminus of co-chaperones
Hop/Sti1p and Hip. Tic40 has been shown to
function at the same stage of import as Tic110
and Hsp93.
As a first step toward investigating the
molecular mechanism of protein translocation
into the chloroplast stroma, we tested if Tic40
directly interacted with Tic110. In vitro pulldown assays indicate that the Tic40 TPR binds
Tic110 (Fig. 1). We next investigated if interaction of Tic40 with Tic110 would affect the interaction of Tic110 with precursors. When Tic110
was preloaded with precursor proteins, the
amount of Tic40 associated with Tic110
Significant Reserach Achievements of Academia Sinica
Most proteins in chloroplasts are encoded
by the nuclear genome and synthesized in the
cytosol as precursors with N-terminal targeting
signals called transit peptides. Precursor import
into chloroplasts is mediated by a protein translocon, which is composed of the Toc (translocon at
the outer envelope membrane of chloroplasts)
and the Tic (translocon at the inner envelope
membrane of chloroplasts) proteins and stromal
chaperones. During import, transit peptides of
precursors first interact with the Toc and then the
Tic proteins. When sufficient ATP is present in
the stroma, precursors are translocated across the
inner envelope membrane into the stroma, and
the transit peptide is removed by the stromal processing peptidase during the translocation.
Although many Tic and Toc proteins have been
identified, the interactions among the translocon
components and the mechanistic steps of the
import process are largely unknown.
73
Life
Sciences
Institute of Molecular Biology
【Fig 1】Tic40 TPR domain binds to Tic110. GST fused
to the entire Tic40 stromal soluble domain (GSTatTic40S), the TPR subdomain (GSTatTic40TPR), or the Hip/Hop subdomain (GSTatTic40Hip/Hop), or GST itself was incubated
with atTic110S-His 6. Only GST-atTic40S and
GST-atTic40TPR pulled down atTic110S-His6.
【Fig 3】Tic40 causes the release of bound transit
peptides from Tic110. (A) atTic110S-His6
was preloaded with a 3H-labeled transit
peptide. An equal amount, or 10-fold or 20fold excess of GST control protein or GSTatTic40S, was then added. Radioactivity in
the supernatant was measured. (B) Protein
import time course into wild-type and tic40mutant chloroplasts.
【Fig 2】Precursors increase the affinity of Tic110 to
Tic40. atTic110S-His6 was premixed with buffer
(lane 1), mature protein marker (RBCS, lane 2),
or precursor protein marker (prRBCS, lane 3).
GST-atTic40S was then added to pull down
atTic110S-His6.
Significant Reserach Achievements of Academia Sinica
74
increased (Fig. 2). However, when an increasing
amount of Tic40 was added to the Tic110-transit
peptide complex, an increasing amount of transit
peptides was released by Tic110 (Fig. 3A). These
results suggested that transit-peptide binding by
Tic110 recruits Tic40 binding to Tic110, which in
turn causes the release of transit peptides from
Tic110. If binding of Tic40 causes the release of
transit peptides from Tic110 in vivo, then in the
absence of Tic40, as in a tic40 null mutant, the
release of transit peptides from Tic110, and therefore the processing of transit peptides, should be
delayed. We therefore compared the import of
precursors into isolated Arabidopsis wild-type
and tic40-1 mutant chloroplasts. The amount of
precursor proteins associated with the membranes was the same in the mutant and the wildtype chloroplasts. However, the appearance of
processed mature proteins was greatly delayed in
the mutant chloroplasts (Fig. 3B).
Since Hsp93 is the only chaperone molecule stably associated with the entire translocon
complex, its ATPase activity should be important
for precursor translocation. We tested whether
transit peptides (TP), Tic110 or different domains
of Tic40 could stimulate the ATPase activity of
Hsp93. Interestingly, it was the Tic40 Hip/Hop
domain, not the TPR domain that had a clear
stimulatory effect on Hsp93 ATP hydrolysis.
We next investigated if the nucleotide state
of Hsp93 affected its association with Tic40.
Hsp93 was pre-incubated with ATP, ADP or the
non-hydrolysable ATP analogue AMP-PNP. The
amount of Hsp93 recovered with Tic40 was higher in the presence of ATP or AMP-PNP, and
lower in the presence of ADP (Fig. 4B), suggesting that Hsp93 may associate with Tic40 in its
Life
Sciences
ATP state and dissociate from Tic40 after ATP is
hydrolyzed to ADP. This result also suggests that
Tic40 most likely functions in stimulating Hsp93
ATPase activity instead of facilitating ATP/ADP
exchange.
【Fig 4】Tic40 Hip/Hop domain activates Hsp93
ATPase activity. (A) His6-Hsp93 was incubated with ATP or ATP plus various other
proteins, as indicated at the bottom of the
graph. The amount of Pi hydrolyzed by
Hsp93 incubated with other proteins was
divided by the amount of Pi hydrolyzed by
Hsp93 alone. (B) His6-Hsp93 was incubated with ATP, ADP and AMP-PNP. GSTatTic40S was then added to pull down
His6-Hsp93.
Based on the data presented above, a model
for the sequential steps of protein translocation
into the chloroplast stroma is presented (Fig. 5):
As the transit peptide of a precursor protein
emerges from the inner envelope membrane
channel, it is bound by the N-terminal part of the
Tic110 stromal domain (Fig. 5A). This binding
causes a conformational change in Tic110 and
recruits Tic40TPR binding to Tic110 (Fig. 5B).
Binding of Tic40TPR to Tic110 causes release of
the transit peptide from Tic110, freeing the transit
peptide for cleavage by the stromal processing
peptidase. Binding of Tic40TPR to Tic110 also
unshields the Tic40 Hip/Hop domain, which then
stimulates ATP hydrolysis by Hsp93 (Fig. 5C).
The energy of ATP hydrolysis by Hsp93 is most
likely used to translocate the processed mature
protein into the stroma (Fig. 5D). Under normal
growth conditions in the light in which the stromal ATP concentration is high, Hsp93 may soon
be reloaded with ATP and be ready for the next
round of precursor translocation.
The original paper was published in The Journal of
Cell Biology 175 (2006): 893-900.
Significant Reserach Achievements of Academia Sinica
【Fig 5】Model for sequential steps of protein translocation into the chloroplast stroma.
75