BIOL 266 – CELL BIOLOGY
Lecture 8:
Protein sorting to organelles (I)
A typical mammalian cell contains up to 10,000
different kinds of proteins
For a cell to function properly, each of its
numerous proteins must be localized to the
correct membrane-bound organelle:
Proteins: Must go to:
Na+/K+ ATPase Plasma membrane
RNA polymerase Nucleus
Proteases Lysosomes
Catalase Peroxisomes
ATP synthase Mitochondria
Hormones Extracellular space
Proteins synthesized in eukaryotic cells
A few proteins: Most of the proteins:
Are encoded by the DNA Are encoded by nuclear DNA
present in mitochondria and
chloroplasts
Are synthesized on ribosomes
in the cytosol
Are synthesized on ribosomes
inside mitochondria and
chloroplasts Are delivered to the organelle
of destination from the
cytosol
Are incorporated directly into
compartments within
mitochondria and chloroplasts
Protein sorting – the process of directing each newly made
protein to a particular membrane-bounded organelle
mRNA mRNA
Proteins are synthesized on
ribosomes in the cytosol
Protein A Protein B
Proteins A and B are sorted
to different organelles
Organelle A Organelle B
Sorting signal – a continuous stretch of 3-60 amino
acids that directs a protein to the organelle in which
it is required
Sorting signal = signal sequence
protein A protein B
signal sequence A signal sequence B
signal sequence A signal sequence B
directs (sorts) protein directs (sorts) protein
A to the organelle A B to the organelle B
Organelle A Organelle B
Nucleus: -Lys-Lys-Lys-Arg-Lys- Peroxisome: - Ser-Lys-Leu-
+ + + + + Polar + Hydrophobic
Role of signal sequences in protein sorting
Proteins destined for the ER possess an amino-terminal signal
sequence that directs them to that organelle
Proteins destined to remain in the cytosol lack this sequence
Cytosolic protein
(no signal sequence)
ER Cytosol
ER protein ER signal sequence
Role of signal sequences in protein sorting
An ER signal sequence is attached to a cytosolic protein
The signal sequence is removed from an ER protein
Result: in each case the altered protein ends up in an abnormal
location in the cell
Conclusion: the ER signal sequence is both necessary and sufficient to
direct a protein to the ER
ER Cytosol
Cytosolic protein with ER ER protein with signal
signal sequence sequence removed
3 consecutive steps in protein sorting:
signal sequence
1 Recognition of the signal sequence
by a shuttling cytosolic receptor
RECEPTOR signal sequence
2 Targeting to the outer surface
of the organellar membrane
RECEPTOR signal sequence
Import: insertion of the targeted protein
3 into the membrane or transport of the
protein across the membrane
A general problem for protein import into organelles:
How to transport the protein across membranes that
are normally impermeable to hydrophilic molecules
Protein:
hydrophilic (contains charged and polar amino acids)
RECEPTOR signal sequence
Import: insertion of the targeted protein
3 into the membrane or transport of the
protein across the membrane
Membrane (lipid bilayer):
Normally impermeable to hydrophilic (charged + polar) molecules
Three main mechanisms by which membrane-bounded
organelles import proteins
nucleus
1 Transport through nuclear pores
Proteins synthesized on
ribosomes in the cytosol
chloroplast
2 Transport across membranes
mitochondrion
peroxisome
ER
Golgi
apparatus
3 Transport by vesicles
nucleus
1 Transport through nuclear pores
Proteins are sorted from the cytosol to
the nucleus
chloroplast
mitochondrion Proteins are transported through the
nuclear pores that function as selective
peroxisome gates, which actively transport only
ER
specific proteins
Golgi
apparatus
Proteins remain folded during the
transport
folded unfolded
nucleus Proteins are sorted from the cytosol to
the ER, mitochondria, chloroplasts or
peroxisomes
Proteins are transported across the
organelle membrane by protein
translocators located in the membrane
chloroplast
2 Transport across membranes
mitochondrion
peroxisome
ER
The transported protein must unfold
Golgi in order to snake through the
apparatus membrane
unfolded folded
translocator
nucleus Proteins are sorted from the ER onward
and from one compartment of the
endomembrane system to another
Proteins are transported by transport
vesicles
Transport vesicles become loaded with a
chloroplast
cargo of proteins from the interior space
(lumen) of one compartment, as they pinch
mitochondrion off from its membrane
These transport vesicles subsequently
peroxisome
ER discharge their cargo into a second
compartment by fusing with its membrane
Golgi
apparatus
3 Transport by vesicles
Proteins remain folded during the transport
Pinches off Fuses
Folded protein Transport vesicle
outer membrane
perinuclear
space inner membrane
nuclear pore nuclear nucleolus chromatin rough endoplasmic
complex lamina reticulum
The nuclear envelope consists of:
Two concentric membranes, called the inner and outer nuclear membranes
The nuclear lamina, a fibrous meshwork that provides structural support to the nucleus
The nuclear pore complexes, the only channels through which molecules are able to
travel between the nucleus and the cytoplasm
The nuclear pore complex (NPC) selectively transports
macromolecules across the nuclear envelope
Large polar molecules (amino
acids, nucleotides, sugars)
Ions
Globular proteins up to 60 Large proteins (> 60 kDa,
kDa (~ 1 nm in diameter) 1-25 nm in diameter)
Diffuse
through water- Do not
filled channels Diffuse
in the NPC
Nuclear pore complex: made up of multiple copies of ~ 100 different proteins
CYTOSOL Cytosolic filament The nuclear pore
Central plug complex (NPC):
Cytosolic ring
The NPC nuclear
ring supports eight
Outer
nuclear ~ 100 nm-long
membrane basket filaments
whose distal ends
Nuclear are joined by the
envelope
terminal ring,
Inner forming a structure
nuclear called the nuclear
Nuclear membrane basket
ring
Basket Nuclear
filament basket
Terminal
ring NUCLEUS
CYTOSOL Cytosolic filament The nuclear pore
Central plug complex (NPC):
Cytosolic ring
The cytosolic ring
supports eight ~ 50
Outer
nuclear nm-long cytosolic
membrane filaments
Nuclear
envelope
Inner
nuclear
Nuclear membrane
ring
Basket Nuclear
filament basket
Terminal
ring NUCLEUS
CYTOSOL Cytosolic filament The nuclear pore
Central plug complex (NPC):
Large proteins (up to
Cytosolic ring
25 nm in diameter)
cannot diffuse
through the water-
Outer
nuclear filled channels (~ 1
membrane nm in diameter) in
the NPC
Nuclear
envelope These proteins are
actively transported
Inner through the central
nuclear plug of the NPC
Nuclear membrane
ring central plug
Basket Nuclear
filament basket
Terminal
ring NUCLEUS water-filled channels
Large proteins cannot pass through the central
plug of the NPC unless they carry an appropriate
signal sequence
This signal sequence, called a nuclear localization
signal (NLS), directs a protein from the cytosol
into the nucleus
NLS: -Lys-Lys-Lys-Arg-Lys-
+ + + + +
The mechanism of active protein transport through the NPC
cargo protein STEP 1
NLS
GTP nuclear import receptors In the cytosol,
GDP nuclear import
1 cytosolic filaments receptors
(importins) bind
to the NLS of the
cargo protein to
be transported
Cytosol
This binding is
driven by the
Nucleus energy provided
by GTP
hydrolysis
The mechanism of active protein transport through the NPC
cargo protein
NLS
GTP nuclear import receptors
STEP 2
GDP
1 cytosolic filaments
Importins help
2 direct the cargo
protein to the
NPC by
interacting with
Cytosol the cytosolic
filaments of the
Nucleus NPC
The mechanism of active protein transport through the NPC
cargo protein STEP 3
NLS
nuclear import receptors The binding of
GTP
the cargo protein
GDP
1 cytosolic filaments to the NPC opens
the pore, and the
cargo protein,
2 with its bound
importins is
transported into
the nucleus
3 Cytosol
ATP Nucleus This transport
ADP process is driven
by the energy
provided by ATP
hydrolysis
The mechanism of active protein transport through the NPC
cargo protein
NLS STEP 4
GTP nuclear import receptors
GDP In the nucleus,
1 cytosolic filaments the “importins-
cargo protein”
complex
2
dissociates
3 Cytosol The importins
are then exported
back through the
ATP Nucleus
ADP
NPC into the
cytosol for reuse
4
Organization of mitochondria
Mitochondria are surrounded by a double-membrane system, consisting
of inner and outer mitochondrial membranes separated by an
intermembrane space
The inner membrane forms numerous folds (cristae), which extend into
the interior (or matrix) of the organelle. Its surface area is substantially
increased by its folding into cristae
cristae
inner membrane
outer membrane
intermediate
space
matrix 1 µm
nucleus Proteins are sorted from the cytosol to
the ER, mitochondria, chloroplasts or
peroxisomes
Proteins are transported across the
organelle membrane by protein
translocators located in the membrane
chloroplast
2 Transport across membranes
mitochondrion
peroxisome
ER
The transported protein must unfold
Golgi in order to snake through the
apparatus membrane
unfolded folded
translocator
Protein import into the mitochondrial matrix
newly synthesized cytosolic precursor protein STEP 1
matrix-targeting (sorting) sequence Cytosolic chaperones
Hsp70 MSF (mitochondrial-
MSF import stimulating
factor) and Hsp70
(heat-shock protein
MSF Hsp70 70):
ATP ATP (1) Use the energy of
MSF Hsp70 ATP hydrolysis to keep
ADP ADP newly synthesized
cytosolic proteins in an
unfolded, import-
competent state
Outer membrane
(2) Deliver unfolded
precursor proteins to
channel-linked
channel
receptors in the outer
channel-linked receptors mitochondrial
membrane
Protein import into the mitochondrial matrix
STEP 2
(1) After chaperones
Outer membrane are released, a
precursor protein
contact site passes through the
channel in the outer
Inn membrane and another
er m channel in the inner
emb membrane
ra n
e
(2) Translocation into
the matrix occurs only
at “contact sites” where
the outer and inner
membranes are in close
proximity
Protein import into the mitochondrial matrix
STEP 3
(1) The newly imported
Outer membrane protein binds to the
matrix chaperone
contact site Hsp70
(2) Hsp70 uses the
Inn energy of ATP
er m
emb hydrolysis to assist
ra n import (to pull the
e
Hsp70 ATP protein inside) into the
Pulling
matrix and to prevent
inside ADP aggregation or
premature folding
Protein import into the mitochondrial matrix
Outer membrane
contact site
STEP 4
Inn
er m After matrix Hsp70 is
emb
ra n released, the matrix-
e
targeting (sorting)
sequence is removed by
Hsp70 a matrix protease
Matrix protease
Protein import into the mitochondrial matrix
STEP 5
Within the matrix:
Outer membrane (1) Some proteins fold
into their mature,
contact site active conformation
without the aid of a
Inn chaperone
er m
emb (2) Other proteins bind
ra n to the chaperone Hsp60
e
(heat-shock protein
ADP 60), which assists in the
ATP
final folding in a
process that requires
Hsp60
energy derived from
ATP hydrolysis
Folded, functionally active proteins
Three-dimensional reconstruction of a region of the smooth and rough ER
Ribosomes are attached to the
cytosolic surface of the rough ER smooth ER
The rough ER
forms oriented
stacks of flattened
cisternae
The smooth ER
membrane is
connected to these
cisternae and
forms a fine
network of tubules
ER lumen
Unlike the proteins that enter the nucleus, mitochondria, chloroplasts and
peroxisomes, most of the proteins that enter the ER begin to be translocated
(transported) across the ER membrane before the protein is completely synthesized
This requires that the ribosome synthesizing the protein is attached to the ER
membrane
Ribosomes are attached to the
cytosolic surface of the rough ER
These membrane-
bound ribosomes
coat the surface of
the ER, creating
regions termed
rough ER because
the characteristic
beaded
appearance when
viewed in an
electron
microscope
ER lumen
There are two separate populations of ribosomes in the cytosol:
(1) membrane-bound ribosomes are attached to the cytosolic surface of the ER
membrane and are synthesizing proteins that are translocated into the ER
(2) free ribosomes are unattached to any membrane and are synthesizing all of
the other proteins
mRNA encoding a cytosolic protein free polyribosome that is synthesizing
remains free in the cytosol cytosolic proteins or proteins imported to
the nucleus, mitochondria and chloroplasts
common pool of ribosomal
subunits in the cytosol
ER membrane
mRNA encoding a protein
Polyribosome bound to the
targeted to the ER by the ER
ER membrane by multiple
targeting sequence remains
growing polypeptde chains
bound to the ER membrane
The translocation of a soluble protein across the ER membrane
into the lumen
STEP 1
mRNA ribosome A cytosolic protein, the
signal recognition
particle (SRP), binds to
the exposed ER signal
sequence and to the
ribosome, thereby
SRP slowing protein
synthesis by the
ribosome
signal sequence
growing polypeptide chain
The translocation of a soluble protein across the ER membrane
into the lumen
STEP 2
The SRP-ribosome
complex then binds to
an SRP receptor in the
ER membrane
SRP
ER membrane
SRP receptor
The translocation of a soluble protein across the ER membrane
into the lumen
STEP 3
As SRP and its receptor
dissociate from the ribosome-
SRP bound polypeptide chain:
(1) The ER signal sequence
binds to the translocon, a
protein translocation channel
in the ER membrane
(2) The ER signal sequence
and the adjacent segment of
the growing polypeptide chain
then insert as a loop into the
central cavity of the
translocon
SRP receptor Translocon Loop of the ribosome-bound
polypeptide chain
The translocation of a soluble protein across the ER membrane
into the lumen
STEP 4
The ER signal sequence is
cleaved by a signal peptidase
in the ER lumen
Signal peptidase
The translocation of a soluble protein across the ER membrane
into the lumen
STEP 5
The ER chaperone protein Hsp70 uses
release of the ribosome
the energy of ATP hydrolysis:
(1) to pull the protein inside, into the
ER lumen
(2) to assist in the final folding of the
translocated protein into its mature,
active state
Pulling Hsp70 ATP
inside ADP Hsp70 ATP
ADP
Folding
