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Biochemistry 4
Intracellular Compartments and Protein Sorting
Mitochondria & Chloroplasts:
organelles, transport to and from
Dr Julia von Blume (MPI Biochemistry)
Prof. Roland Beckmann
Dr. Thomas Becker
Literature
Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter
Essential Cell Biology 3rd edition, 2010, Garland Science
Alberts, Johnson, Lewis, Raff, Roberts, Walter Molecular
Biology of the Cell 5th edition, 2008, Garland Science
Eurkaryotic cells are compartmentalized
Features of cellular compartmentalization
1. cellular compartments are enclosed by a membrane
2. each compartment is endowed with a characteristic set of enzymes
3. each compartment contains specialized molecules
4. each compartment is equipped with specialized transport systems
Eurkaryotic cells are compartmentalized
protein synthesis and
degradation
ATP generation
host of the DNA
gene expression regulation
degradation of
cellular material
protein synthesis
lipid production
Ca2+ store
protein sorting
lipid sorting
distribution of proteins
protein modification
protein sorting
The synthesis of all proteins starts in the cytosol
Room1
Room II
Room III
How are proteins within cells targeted to
their specific location within the cell?
How are proteins within a cell targeted
to a specific compartment?
Proteins have intrinsic signals that govern their
transport and localization in the cell
Protein
signal sequence (ss)
Günther Blobel, Nobel Prize for Physiology or Medicine, 1999
Proteins are targeted to specific
compartments by signal sequences
-  proteins are guided to correct cellular compartment by a signal sequence
- recognition follows the biophysical properties of the sequence
N-terminal (cleaved or uncleaved)
C-terminal (uncleaved)
internal (uncleaved)
(positively charged amino acids)
(hydrophobic amino acids)
(hydroxylated amino acids)
(negatively charged amino acids)
resides in the cytosol. After the cDNA encoding this
protein is transfected into cells, the location of the
fusion protein is determined by immunostaining or
by cell fractionation.
in the in vitro
Three method
translocated in
Approach to define signal sequences
signal sequence a or b
gene encoding cytosolic protein
1. The labeled
with the organ
plasmid used to transfect cells
A
A
B
signal sequence a (
)
directs fusion protein
to organelle A
B
signal sequence b (
)
directs fusion protein
to organelle B
By altering the signal sequence using site-directed
mutagenesis, we can determine which structural
3. The protein
digestion whe
added to the
but is suscepti
first added to
organelle mem
How does protein transport in eukaryotic
cells work?
There are three major protein transport principles
for entry/transport between different
compartments
1. gated transport
2. transmembrane transport
3. vesicular transport
In gated transport proteins move
between cytosol and nuclear pore complex
- proteins move between cytosol and nucleus
(topologically equivalent)
- proteins move through the nuclear pore
complex
nuclear pore complex acts as gate that actively
transports:
- specific macromolecules
- macromolecular assemblies
nuclear pore complex also allows diffusion of
smaller molecules.
In transmembrane transport proteins are
actively translocated across the membrane
- transmembrane protein translocators move
proteins across a membrane from the cytosol
- protein must unfold to snake through the
tanslocator
transport from cytosol to the:
endoplasmic reticulum lumen
mitochondria
chloroplasts in plant cells
Wickner and Schekman, Science, 2005
In vesicular transport membrane enclosed
vesicles transport proteins between different
compartments
- membrane enclosed transport intermediates (transport vesicles) ferry proteins from one
compartment to the other
- vesicles become loaded with cargo molecules derived from the lumen of the
compartment as they bud and pinch off from its membrane
- cargo becomes discharged into a second compartment by fusing with the membrane
enclosing that compartment
Function of mitochondria and
chloroplasts
photosynthesis
electron transport
oxidative phosphorylation
Mitochondria and chloroplasts are
derived from Gram-negative bacteria
•  Prokaryotic cells have no internal membranes
•  Plasma membrane provides all membrane-dependent functions:
Pumping of ions, ATP synthesis, protein secretion, lipid synthesis
Mitochondria and chloroplasts are
derived from Gram-negative bacteria
outer membrane:
- beta-barrel proteins
- mildly hydrophobic
peptidoglycan
inner membrane:
- alpha-helical proteins
- highly hydrophobic
Mitochondria and chloroplasts are
endosymbiotic organelles
•  eukaryotic cells started out as anaerobic
organisms without mitochondria/chloroplasts
•  established a stable endosymbiotic relation with
a bacterium whose oxidative phosphorylation
system they subverted for their own use
Hypothesis of the evolutionary origins of
mitochondria and chloroplasts
prokaryotic
Accumulating biochemical data suggest that
- the double membrane enclosing chloroplasts and mitochondria derived from the ancestral bacteria
- the phagosomal membrane disappeared during organelle evolution
Structure and function of mitochondria
- double membrane enclosed organelles
- specialized on ATP-Synthesis using energy
derived from electron transport and oxidative
phosphorylation
two subcompartments:
matrix space and intermembrane space
membranes:
outer and inner membrane
- contain DNA and ribosomes + other
components required for protein
synthesis
- most of the mitochondrial proteins are
encoded in the cell nucleus
- most proteins have to be imported from the
cytosol into the mitochondria
Structure and function of chloroplasts
- double membrane enclosed organelles
- specialized on ATP-Synthesis
derived from photosynthesis
three subcompartments:
intermembrane space, stroma,
thylakoid space
membranes:
outer, inner and thylakoid membrane
(contains photosynthetic pigments)
- contain DNA and ribosomes + other
components required for protein
synthesis
- most of the chloroplast proteins are encoded
in the cell nucleus
Mitochondria and chloroplasts have transferred
most of their genome into the nucleus
most of the genes encoding
mitochondrial or chloroplast proteins
are encoded by nuclear genes
requirement for import machineries
mitochondrial genome encodes:
- a few inner membrane proteins
involved in electron transport reactions
- ribosomal RNAs
- some tRNAs
How does the protein transport into
mitochondria work?
Proteins destined to matrix:
N-terminal signal sequence
Cleaved by signal peptidase
Proteins destined to outer, many
intermembrane and inner
membrane proteins:
internal signal sequence
Signal sequence: not cleaved
How are the proteins encoded by nuclear
genes transported to mitochondria/
chloroplasts?
Translocation into mitochondria depends on
signal sequences and protein translocators
complexes contain proteins that act as
a) receptors for mitochondrial precursor proteins
b) proteins that form translocation channels
Signal sequences of mitochondrial proteins are
necessary and sufficient for import and localization
N-terminal signal sequence for import into mitochondria
hydrophobic and and positively charged amino
acids fold into an amphiphilic alpha helix
front side hydrophilic
back side hydrophobic
binds into hydrophobic groove of the TOM20 receptor
translocation
Approach to test the significance of a signal
sequence for mitochondrial import
isolated
mitochondria
signal sequence
is cleaved off
TOM and SAM complex translocate mitochondrial
proteins into the outer mitochondrial membrane
TOM complex:
(translocase of the outer mitochondrial membrane)
- import of all nucleus-encoded mitochondrial proteins
- transfers proteins across the outer membrane
- helps with the insertion of transmembrane
proteins in outer membrane
ß-barrel proteins are passed to the SAM complex that
helps with folding
The TIM23 complex transports soluble
proteins into the matrix and helps to
insert proteins into the inner membrane
TIM23 complex:
(translocase of the inner mitochondrial membrane)
transfers soluble proteins into the matrix
helps with the insertion of transmembrane proteins in
inner membrane
complex spans two membranes
one of the core components of the complex contains a
hydrophobic alpha-helical extension that is inserted in the
outer mitochondrial membrane
complex is associated to mitochondrial Hsp70 on the matrix
side, which acts as an import ATPase
(ATPase hydrolyzes ATP to pull proteins through the pore)
The TIM22 complex mediates the insertion of
a subclass of inner membrane proteins
TIM22 complex:
INNER MITOCHONDRIAL MEMBRANE
(translocase of the inner mitochondrial membrane)
helps with the insertion of a subclass of inner membrane proteins
inculding:
transporters that move ATP/ADP and Phosphate in and out of
the mitochondria
MATRIX
The OXA complex mediates the insertion of inner
membrane proteins synthesized by mitochondria
OXA complex:
mediates insertion of inner membrane proteins that are
synthesized within the mitochondria
helps to insert some imported inner membrane proteins
that are initially transported to into the matrix space by
other complexes
Mitochondrial protein import
Which energy sources drive the directional
transport of the precursor proteins across
the mitochondrial membranes?
two different energy sources:
ATP hydrolysis
membrane potential across the inner mitochondrial membrane
ATP hydrolysis and a membrane potential
drive protein import into the mitochondria
Import to the mitochondria requires ATP hydrolysis:
a) outside the mitochondria
release of the unfolded protein from Hsp70
proteins requires ATP hydrolysis
b) import into the matrix by TIM23
mitochondrial Hsp70 pulls the precursor protein
through the translocation channel
release of the unfolded protein from mitochondrial
Hsp70 requires ATP
ATP hydrolysis and a membrane
potential drive protein import into the
mitochondria
translocation of the transported precursor protein through the TIM channel requires
membrane potential (H+gradient across the inner mitochondrial membrane)
the signal sequence of the precursor protein is translocated into the matrix space in a
process that requires a membrane potential across the inner membrane
ATP hydrolysis and a membrane potential
drive protein import into the mitochondria
How does mitochondrial hsp70 drive import?
Two models:
trapped in matrix
back and forth sliding
active pulling of hsp70
Gram negative bacteria and mitochondria
insert their beta barrel proteins in a similar way
One of the central units of the SAM complex is homologous to the
bacterial outer membrane protein that helps to insert beta-barrel
proteins into the bacterial outer membrane from the periplasmic space
conserved pathway if inserting of beta barrel proteins is further
evidence for the endosymbiotic theory
How are proteins inserted into the
intermembrane space and inner membrane
of mitochondria?
Transport into the inner mitochondrial membrane
and intermembrane space occurs via similar routes
Insertion of proteins into the inner membrane:
TOM
TIM23
Transport into the inner mitochondrial membrane
and intermembrane space occurs via similar routes
Insertion of proteins into the inner membrane:
Transport into the inner mitochondrial membrane
and intermembrane space occurs via similar routes
Insertion of proteins into intermembrane space:
Transport into the inner mitochondrial membrane
and intermembrane space occurs via similar routes
Insertion of multispan membrane proteins:
Mitochondria are the sites of ATP synthesis: proteins + small metabolites required
outer membrane is permeable, inner membrane is impermeable
metabolite-specific transporters required for transport of small molecules across the inner membrane
transporters of inner membrane are multipass membrane proteins without cleavable signal sequence
How does the protein transport into chloroplasts
work?
Thylakoids are membrane enclosed compartments
Photosynthetic system
ATP synthase
Two signal sequences direct proteins to
the thylakoid membrane in chloroplasts
precursor proteins have a hydrophobic
thylakoid signal sequence
followed the N-terminal
chloroplast signal sequence
1. proteins pass across the double
membrane into the stroma
across outer membrane: TOC
across inner membrane: TIC
2. translocation to the thylakoid
membrane or into thylakoid space
There are four routes of protein translocation
across/into the thylakoid membrane
1. Sec pathway: uses components that are homolog to the Sec proteins in bacteria which mediate the
translocation across the bacterial plasma membrane
2. SRP-like pathway: uses the homolog of the signal recognition particle, or SRP
3. TAT pathway: twin arginine translocation, requires H+ for translocation
4. spontaneous insertion pathway: does not need any protein translocation
Protein transport into chloroplasts mostly
resembles transport into mitochondria
Similarities between protein transport to mitochondria and chloroplasts
1. import of most proteins occurs post translationally
2. use separate translocation complexes in each membrane
3. energy requirement
4. use of amphiphilic N-terminal signal sequences that are removed after use
Differences in mitochondrial and
chloroplast protein transport
Differences in mitochondrial and chloroplast protein transport
1. subcompartments
mitochondria, 2; chloroplasts 3
2. protein components for import differ
3. chloroplasts use GTP- and ATP hydrolysis to power the transport of proteins across their
inner membrane whereas mitochondria use membrane potential
4. chloroplasts use H+gradient for transport across the thylakoid membrane
5. signal sequences are different because plant cells have chloroplasts and mitochondria
Take home message
• 
although mitochondria and chloroplasts have their own genetic systems they produce
only a small proportion of their own proteins
• 
chloroplasts and mitochondria import most of their proteins from the cytosol using
similar import mechanisms
• 
proteins are transported in an unfolded state across outer and inner membrane
simultaneously into the matrix space or stroma
• 
Both ATP hydrolysis and membrane potential across the inner membrane drive
translocation into mitochondria, whereas GTP and ATP hydrolysis drive
translocation into chloroplast
• 
Chaperones of the Hsp70 family keep the protein in a unfolded state in the cytosol
or mitochondrial Hsp70 chaperones pull the polypeptide chain into the matrix or
stroma
• 
Translocation requires a signal sequence (N-terminal, internal)
• 
Import into the thylakoid membrane can occur by several routes depending on the
chaperones and energy source used.