Translocation into mitochondria/plastids/peroxisome
1. The question and complexity
Both mitochondria and plastids have their own genome but a large
number of proteins in these organelles are encoded in the
nucleus, made in the cytosol, and eventually cross the organelle
membrane into the right compartment to do their job.
Complexity: look at the complexity of these organelles—there are
at least 6 different sub-organelle space in plastid/chloroplast
and 4 in the mitochondria
The coordination of
Protein targeting:
Both plastid-made
and cytosol-made
proteins need to go
to 6 spaces inside
the chloroplasts!
How do they know
where to go and
what are the
signals?
2. Signal sequences and receptors in chloroplast targeting
1) Signal sequence of chloroplast proteins is called transit
peptide
A precursor made in the cytosol contains N-terminal sequence that
is required and sufficient for chloroplast targeting. After
translocation, the transit peptide is cleaved by a peptidase.
required and sufficient: If you delete the sequence off the
chloroplast proteins, these proteins can not be transported into
chloroplasts anymore; if you attach the transit peptide to
other non-chloroplast proteins, then the sequence can direct
these “foreign” proteins into chloroplasts.
2) How do you measure chloroplast import?
Chloroplasts and
protein substrates:
chop the leaves
and isolate
chloroplasts by
centrifugation. The
protein substrate
can be made by in
vitro transcription
and translation in
the presence of
Met-S-35 to label
the proteins
radioactive.
The re-purification of
chloroplast and
treatment with
protease (why?).
The gel picture: lane 1 is the precursor substrate before
mixing with the chloroplasts, lane 2 contains the proteins
from chloroplasts without protease treatment; lane 3 is
the protein after protease treatment of the chloroplasts.
Small subunit of Rubisco as example.
More recent studies using fusion proteins: fuse certain protein with
an easy-to-detect markers such as GFP (green fluorescence protein)
or GUS. Transform the constructed gene fusion into the plants to
produce transgenic plants or do the transient assay but
bombardment of t he gene into a plant tissue---measure the marker
protein location. One can fuse just the transit peptide to a marker
and see this sequence direct the transport of the marker into the
chloroplast.
Ssu protein
Ssu transit peptide-fused to GUS
Ssu transit peptide-fused to GFP
3) Chloroplast proteins are delivered across the envelope through protein
channels
a) The chaperones hsp70 type I helps to keep the proteins unfolded
before delivery through the channel.
b) Several proteins inside the outer and inner membrane envelope form
a “channel’-like structure and help substrate proteins go through the
double membranes. The details involved in the interaction between
the substrate and channel proteins are not understood. The outer
membrane proteins called TOC (translocon of outer chloroplast
envelope) and the inner membrane proteins are called TIC
(translocon of inner chloroplast envelope).
c) Both ATP and GTP are consumed.
d) Once delivered into the stroma, a peptidase cleaves the transit
sequence off the precursor.
e) The proteins are then folded by chaperones (type I or type II).
f) Proteins are assembled into complexes if needed.
Across the double envelope membranes
4) The signal for thylakoid targeting requires two regions
of N-terminal sequence---a bipartite transit sequence
Was discovered by study of plastocyanin, a thylakoid
lumen protein (in the electron transport chain between the
PSII and PSI). A copper-binding protein that appears
bright blue color.
Envelope/thylakoid
1
2
3
stroma
4
P
I
m
thylakoid
5) Trans-thylakoid pathways
At least three different mechanisms are involved in protein
targeting into thylakoid lumen/membrane. Different pathway
help different substrate proteins. The “signals” for each
pathway is not very well understood.
a) Sec pathway--- because this pathway consists of proteins
similar to those in the bacterial secretory pathway. This
pathway is stimulated by pH gradient and ATP (plastocyanin
takes this pathway)
b) SRP pathway---consists of proteins with similarity to the ER
translocation pathway. Stimulated by pH gradient and GTP.
The light harvesting protein takes this pathway to get
integrated into the thylakoid membrane.
c) Delta pH pathway—only requires pH gradient (eg, oxygen
evolution complex protein)
Summary
Where and the how
are proteins
targeted?
3. Protein targeting into the mitochondria---quite similar to
the chloroplast mechanisms:
1) Also involves N-terminal sequences as signals; in fact the
nature of the peptide sequence is also similar to the
chloroplast targeting sequence although they are not
exchangable.
2) Also involve outer and inner membrane proteins as
translocons (they are called TOM and TIM instead of
TOC/TIC).
3) Also requires ATP and chaperone type I (hsp70)
4) Requires more than one sequence signal for targeting to
the intermembrane space; multiple pathways for targeting
5) Also involves peptidase to cleave precursors
6) Also involve chaperone to re-fold the proteins inside the
organelle.
Model of TOM/TIM:
Further enforce the
idea of protein
channels formed by
some of the proteins
in he membranes