Transcript rough ER

Intracellular compartments
Protein Sorting and Transport
Compartments of an Animal Cell
Compartments of an Animal Cell - TEM
Procaryotic Cell
Eucaryotic Cell
Typical Plant Cell
Golgi body
vesicle
central vacuole
rough endoplasm
reticulum (rough ER)
microfilaments
(components of
cytoskeleton)
ribosomes (attached to
rough ER)
ribosomes (free in
cytoplasm)
smooth endoplasmic
reticulum
(smooth ER)
mitochondrion
chloroplast
microtubules
(components of
cytoplasm)
DNA + nucleoplasm
nucleolus
nuclear envelope
plasma membrane
cell wall
NUCLEUS
Animal Cell
Plant Cell
Functions of major intracellular compartments:
•Nucleus - contains main genome, DNA and RNA synthesis.
•Cytosol - most protein synthesis, glycolysis and metabolic pathways
synthesizing amino acids, nucleotides.
•Endoplasmic reticulum - synthesis of membrane proteins, lipid
synthesis.
•Golgi apparatus - covalent modification of proteins from ER,
sorting of proteins for transport to other parts of the cell.
•Mitochondria and chloroplasts (plants) - ATP synthesis.
•Lysosomes - degradation of defunct intracellular organelles and
material taken in from the outside of the cell by endocytosis.
•Endosomes - sorts proteins received from both the endocytic
pathway and from the Golgi apparatus.
•Peroxisomes - oxidize a variety of small molecules.
Three basic modes of transport
1.
Gated transport
2. Transmembrane transport
3. Vesicular transport
Roadmap of protein traffic
The genesis and function of
internal compartments
depends on the appropriate
targeting of proteins
Some of the green route illustrated.
Protein Sorting – Signal Sequences
A simple experiment shows that many sorting signals
consist of a continuous stretch of amino acid sequence
called a “signal sequence”.
Fusing sorting signals to
GFP is particularly good
way to do this
experiment.
GFP
Cytoplasmic
Nuclear
PAX-GFP
Actin-GFP
Signal Sequences
Nuclear Import and Export
•Nuclear envelope
consists of two
concentric lipid bilayers.
•The perinuclear space
is contiguous with the
lumen of the ER.
•Bidirectional transport
occurs through the
nuclear pore complexes.
Nuclear Pore
The nuclear pore complex
is an aqueous channel that
allows diffusion of small
molecules and proteins up
to 60kD. Hence, transport
of such molecules is
passive.
Molecules larger than 9 nm or 60 KDa must have
Nuclear Localization Signals (NLS) and be actively
transported into the nucleus
The Mechanism of Nuclear Transport
In most cases, nuclear
localization of large proteins
relies on a signal sequence
called a Nuclear localization
signal or NLS.
•NLS can be located
anywhere in the primary
sequence of the protein.
•Usually arginine and lysinerich and quite short.
•There are some exceptions
where nuclear localization
relies on a signal patch.
The NLS directs the protein for transport through the
nuclear pore complex, and proteins maintain their
tertiary and quaternary structures during transport.
When gold beads are coated
with the NLS, the beads can be
seen passing through nuclear
pore complexes. The maximum
size bead that can be
transported is 26 nm. Since the
gold bead can’t compress, the
opening of the pore must be able
to expand.
An asymmetric distribution of a Ran-GAP and a Ran-GEF controls
the activity of Ran in a way that allows Ran to mediate active
transport through the nuclear pore complex.
Ran-GAP in the cytosol
causes Ran-GDP to
predominate in the
cytosol.
Ran-GEF in the nucleus
causes Ran-GTP to
predominate in the
nucleus.
Ran: Monomeric GTPase
Ran-GAP: GTPase-activating protein
Ran-GEF: Guanine exchange factor
The NLS associates with soluble cytosolic proteins called nuclear
import receptors. The nuclear import receptors also bind the nuclear
pore complex so they serve to bring the protein containing the NLS
to the nuclear pore complex.
Structure of Ran-GTP & How its binding to import receptor
leads to release of cargo protein
Import process
1. Nuclear import receptor associates
with cargo and brings the cargo to the
nuclear pore.
2. Somehow the receptor/cargo
complex moves through the pore (FGrepeats?).
3. Once in the nucleus, Ran-GTP
displaces the cargo from the
receptor. Ran-GTP is present in the
nucleus because of the Ran-GEF.
4. Receptor/Ran-GTP complex move
through the pore.
5. Once the receptor returns to the
cytosol, Ran-GAP and a Ran binding
protein collaborate to cause Ran to
dissociate from the receptor and
hydrolyze GTP.
The nuclear import players:
•Nuclear import receptor binds the cargo.
•NLS is in the amino acid sequence of the cargo protein.
•Ran-GTP and Ran-GDP are different forms of Ran bound
either to GTP or GDP. Ran-GTP causes the NLS to
dissociate from the Nuclear import receptor.
•Ran-GAP is distinct from Ran but causes Ran to
hydrolyze GTP. Hence, Ran-GAP promotes the conversion
of Ran-GTP to Ran-GDP.
•Ran-GEF is distinct from Ran but causes Ran to release
GDP and bind a different molecule of GTP. Hence, RanGEF promotes the conversion of Ran-GDP to Ran-GTP.
Export of RNA and proteins relies on nuclear export receptors associating
with nuclear export signals (NES) found on proteins and RNA-bound
proteins.
•Nuclear export receptors are structurally similar to nuclear import
receptors.
•Nuclear export receptor bind the nuclear export signals and bring the
protein to the nuclear pore complex for subsequent transport.
•Transport occurs through the same pores through which proteins are
imported from the cytosol.
•Ran regulates the interaction between the export receptor and the “NES”.
The Ran-GTP promotes association of the receptor/cargo complex with the
pore in the nucleus and hydrolysis of the GTP on the cytosolic side causes
the resulting Ran GDP to dissociate the export receptor from its cargo.
Note - nuclear export receptors do not bind directly to RNA, they bind
proteins bound to the RNA.
Transport of Proteins into Mitochondria and Chloroplasts
•Organelles specialized for ATP synthesis.
•Most, but not all, proteins are encoded by the nuclear genome
and synthesized in the cytosol.
•Proteins must be transported to one of multiple membranes or
compartments.
Mit. Import signal
Polar (+) and
non-polar striped
alpha-helix
Protein Transporters of the Mit.
membranes
Import of the protein into the matrix is directed by an N-terminal
signal sequence.
•For polypeptides encoded by the nuclear genome, synthesis of the polypeptide is first
completed in the cytosol. Transport occurs by a posttranslational mechanism.
•Signal sequence at the N-terminus associates with the TOM complex located in the
outer mitochondrial membrane. TOM is both a receptor for the signal sequence and a
translocase.
•The polypeptide is passed from TOM to TIM in the innermembrane. During the
transport process, the polypeptide traverses both inner and outer membranes via the two
translocators at a point known as a contact site.
•The polypeptide is imported and the signal peptide is removed by a signal peptidase.
The energetics of import - import requires ATP hydrolysis and an
electrochemical proton gradient.
•ATP hydrolysis regulates the association of chaperone proteins in the cytosol that serve
to keep the polypeptide in an unfolded state prior to association with TOM.
•Electrochemical proton gradient in the inner membrane draws the signal sequence
through TIM into the matrix.
•Chaperone proteins in the matrix use the energy of ATP hydrolysis to pull the
polypeptide into the matrix and guide proper folding.
The N-terminal signal
sequence for mitochondrial
import is a positively
charged, amphipathic alpha
helix. Hence, the membrane
potential across the inner
mitochondrial membrane may
“electrophorese” the signal
sequence through the TIM
complex.
Protein import into the inner mitochondrial membrane or
intermembrane space
The human mitochondrial genome
Transport into chloroplasts is similar to mitochondria except
there is a 3rd membrane that can be targeted. Targeting the
thylakoid membrane involves a second signal sequence.
In the case of
chloroplasts, the
electrochemical proton
gradient is at the
thylakoid membrane where
this gradient participates
in transport. Transport
across the chloroplast
inner membrane is
powered by GTP and ATP
hydrolysis.
Comparison of mitochondrial and nuclear import.
Nucleus
Mitochondria
Fate of the
signal sequence
Unchanged after
transport
Removed by signal
peptidase
Energy
GTP hydrolysis
ATP hydrolysis and
electrochem ical
proton gradient
Conformation of
the transported
protein
Folded
Unfolded
Signal sequence
Short, positively
charged, located
anywhe re
N-terminus,
amphipathic alpha
hel ix
Peroxisomes (which are not
part of the endomembrane
system) contain enzymes
used to incorporate
hydrogens with oxygen to
make hydrogen peroxide
which is then converted to
water and oxygen. They
function in a variety of
situations including
detoxifying poisons by
transferring hydrogens
from them to oxygen.
The dense bodies within the
peroxisomes are
crystallized enzymes.
Peroxisomes are formed
from phospholipids and
enzymes in the cytosol, not
by pinching off from the
endomembrane system.
Peroxisomes are organelles that perform a variety of oxidation
reactions including ones that breakdown fatty acids and toxic
molecules that enter the cell from the blood stream.
Genetic defects in peroxisomes often cause neurological
problems because a particular lipid found in myelin is produced
in the peroxisomes.
Import of proteins involves short signal sequences. The most
unusual aspect of the transport process is that oligomeric
proteins don’t have to unfold. Very little is known about the
transport process.
Endoplasmic reticulum
Rough ER
Rough ER’s rough
appearance is due to
ribosomes.
ER lumen is one continuous
space that merges with the
perinuclear space.
Smooth ER
Functions of the ER
•Starting point for newly synthesized proteins destined for Golgi,
Endosome, Lysosomes, Secretory vesicles, and the Plasma membrane
(see below).
•Establishes orientation of proteins in the membrane.
•Site of phospholipid and cholesterol synthesis.
•Initiation site for N-linked glycosylation of proteins.
•Sequesters Ca++ - sarcoplasmic reticulum in muscle is a specialized
ER.
Free and Bound Ribosomes
•A signal sequence of
approximately 20 amino acids and
rich with hydrophobic amino acids
is often located at the N-terminus.
•Since the ribosome masks about
30 amino acids, the signal sequence
isn’t fully exposed until the
nascent polypeptide is about 50
amino acids long.
•SRP-ribosome attaches to SRP
receptor and then docks on a
protein translocator.
•SRP and receptor dissociate.
•Translation and translocation
proceed in unison - co-translational
transport.
•The energy for transport is
provided by the translation
process - as the polypeptide grows,
it is pushed through the protein
translocator.
SRP: signal-recognition particle
SRP receptor
The signal sequence of secreted proteins is cleaved by a signal
peptidase. In the literature, the signal sequence of secreted proteins
is often called a “leader peptide”.
Translocation of protein across the ER membrane
A single-pass membrane protein
Co-translational transport must be able to generate a
diverse array of configurations.
For both single-pass and
multipass transmembrane
proteins, some types will
have the N-terminus
projecting into the cytosol
and others will have the Cterminus projecting into the
cytosol.
These two examples have
an internal start-transfer
sequence. In the top case,
the orientation is such that
translation by the
ribosome pushes the
growing chain through the
translocator. In the
bottom case, the
orientation is such that
ribosome separates from
the protein translocator
and chain growth occurs in
the cytosol.
In this case, a start transfer sequence followed by a
stop transfer sequence yields a 2-pass transmembrane
protein with the N and C-terminus in the cytosol.
Things can get pretty complicated!
GPI anchored integral membrane proteins are
generated in the ER.
N-linked glycosylation of ER proteins.
Most of the glycosylation associated with proteins outside the cell
begins in the ER.
A large preformed
oligosaccharide is transferred
from a lipid called dolichol to the
side-chain of asparagine.
Because the covalent linkage is to
the nitrogen of the asparagine
side-chain, this is called N-linked
glycosylation.
In the ER, N-linked
oligosaccharides are modified
and the modifications are used as
signals to distinguish properly
folded from unfolded proteins.
ABO blood type is determined by two
glycosyltransferases
ER protein folding
If a protein doesn’t achieve a properly folded state, it
gets exported from the ER into the cytosol where it is
degraded by the proteasome.
Phospholipids are synthesized in the cytoplasmic leaflet
of the ER.
Phospholipid translocators flip-flop the phospholipids.
Transfer of lipids to other organelles.
Most lipids for other organelles are synthesized at the ER.
•Lateral diffusion will supply the nuclear membrane.
•Vesicular transport will supply organelles in the secretory
pathway and lysosomes (vesicular transport will be described
soon)
•Phospholipid exchange proteins deliver phospholipids to the
mitochondria, chloroplasts and peroxisomes.
Intracellular Vesicular Transport
The Golgi apparatus plays a central role in vesicular traffic
within cells; the post office of the eukaryotic cell.
Vesicular transport delivers components between
compartments in the biosynthetic-secretory and endocytic
pathways.
Note how the cytoplasmic domain of my hypothetical
protein remains in contact with the cytoplasm. Vesicular
transport maintains the membrane orientation.
Once proteins that don’t normally reside in the ER are properly
folded, they are transported to the golgi apparatus.
Proteins exiting the ER join the Golgi apparatus at the
cis Golgi network. The Golgi apparatus consists of a
collection of stacked compartments.
Three coat proteins drive vesicle formation at various
locations in the cell.
A clathrin-coated pit on the cytosolic
face of the plasma membrane
Clathrin associates via adaptins with receptors in the donor
membrane. The receptors bind specific cargo. The clathrin
assembles into a cage that encapsulates a region of membrane.
Then dynamin causes the membrane to pinch off forming a vesicle.
Energy requirements:
•GTP hydrolysis by dynamin accompanies pinching off.
•ATP hydrolysis by chaperone proteins (not shown).
COPII vesicle formation is mediated by a monomeric
GTPase. A GEF in the donor membrane interacts with the
GTPase, Sar1, causing GDP/GTP exchange. Sar1-GTP extends a
fatty acid tail that inserts into the membrane. COPII assembles
on the Sar1 to form a vesicle.
COPI vesicle formation involves a protein called ARF that is analogous
to Sar1.
Targeting of the vesicles is achieved by complementary
sets of v-SNAREs and t-SNAREs.
Conformational changes
in the v-SNARE/tSNARE complex appear
to drive membrane
fusion without ATP or
GTP hydrolysis.
After membrane fusion, ATP hydrolysis is used to pry
apart the v-SNARE/t-SNARE complex.
Transport from the ER through the Golgi Apparatus continuation of the biosynthetic-secretory pathway.
Properly folded proteins
are loaded into COPII
transport vesicles, while
unfolded proteins
remain associated with
chaperones in the ER
until folding is complete.
If a protein doesn’t achieve a properly folded state, it
gets exported from the ER into the cytosol where it is
degraded by the proteosome.
When the protein is properly folded, COPII coated
vesicles transport the proteins via the vesicular tubular
cluster (vtc) to the cis-Golgi network.
•The COPII coating is removed
(Sar1 hydolyzes GTP) and the
vesicles fuse with each other
to form the vtc.
•The vtc is motored along
microtubules that function like
railroad tracks.
•The vtc fuses with the cisGolgi network.
Some proteins exiting the ER are returned to the ER by COPI coated
vesicles. These proteins are identified by the presence of specific
signal sequences that interact with the COPI vesicles or associate
with specific receptors.
Examples of retrieved
proteins:
•v-SNAREs from the
ER.
•ER chaperones like
BiP that are
mistakenly
transported.
Proteins exiting the ER join the Golgi apparatus at the
cis Golgi network. The Golgi apparatus consists of a
collection of stacked compartments.
TEMs of
Golgi
Apparatuses.
In an
animal
secretory cell
and
an algal cell
Chlamydomonas
(trans)
Note convex
cis face and
concave trans face.
The Golgi Apparatus has two major functions:
1. Modifies the N-linked oligosaccharides and adds O-linked
oligosaccharides.
2. Sorts proteins so that when they exit the trans Golgi network,
they are delivered to the correct destination.
Modification of the N-linked oligosaccharides is done by
enzymes in the lumen of various Golgi compartments.
1. Sorting in TGN
2. Protection from protease digestion
3. Cell to cell adhesion via selectins
It is uncertain how proteins move through the Golgi apparatus.
Stationary compartments
with vesicles transporting
between compartments.
Large moving compartments
that mature into the TGN,
and return enzymes to
trailing compartments by
retrieval vesicles.
Cytochemical demonstration of different compartments of Golgi
Unstained
nucleoside
diphosphatase
(trans)
Osmium
Golgi Rxn
(cis)
Acid
phosphatase
(TGN)
One ultimate destination of some proteins that arrive in the TGN is
the lysosome. These proteins include acid hydrolases.
Lysosomes are like the stomach
of the cell. They are organelles
surrounded by a single membrane
and filled with enzymes called
acid hydrolases that digest
(degrade) a variety of
macromolecules. A vacuolar H+
ATPase pumps protons into the
lysosome causing the pH to be
~5.
The macromolecules that are degraded in the lysosome
arrive by endocytosis, phagocytosis, or autophagy.
The acid hydrolases in the lysosome are sorted in the TGN
based on the chemical marker mannose 6-phosphate.
The phosphate
is added in the
Golgi
This was first
attached in the
ER.
Adaptins bridge
the M6P receptor
to clathrin.
Hydrolases are
transported to the late
endosome which later
matures into a lysosome.
Acidic pH causes
hydrolase to dissociate
from the receptor.
Endocytosis is a process by which cells take up substances by
invaginating the plasma membrane. This process can capture both
membrane bound and soluble components.
There are several subclasses of endocytosis:
•Phagocytosis takes up large particles and cells.
•Pinocytosis continuously takes up small amounts of fluid.
•Receptor-mediated endocytosis selectively takes up membrane
receptors and associated ligands.
Endocytosis takes up large amounts of the plasma membrane and is
balanced by the return of membrane components to the plasma
membrane by exocytosis.
Phagocytosis is performed primarily by white blood cells called
Macrophages, Neutrophils and Dendritic cells. These cells receptors
in the plasma membrane to recognize their targets. For example,
macrophages have receptor that recognizes phosphatidylserine which
becomes exposed on the surface of dead cells.
Pinocytosis is performed by clathrin coated pits and by caveolae.
Clathrin coated pits are precursors to clathrin coated vesicles.
Calveolae are deep invaginations in the plasma membrane that are
thought to be formed from lipid rafts (regions high in cholesterol and
glycolipids) and a transmembrane protein called caveolin.
Receptor-mediated uptake of
LDL is one of the best
understood examples of
receptor-mediated
endocytosis. LDL is a
protein-lipid complex that
transports cholesterol-fatty
acid esters in the blood
stream. LDL normally
supplies cholesterol to cells.
Defects in the endocytic
process result in high blood
levels of LDL. High LDL
predisposes individuals for
atherosclerosis.
Normally, the
receptors associate
with adaptin.
Some individuals have
defects in the
cytoplasmic domain
recognized by
adaptin.
Other genetic defects that result in elevated blood
levels of LDL:
•absence of LDL receptor
•defective LDL-binding site in the LDL receptor.
Receptor mediated
endocytosis involves
transmembrane receptors
that bind specific ligands.
The specificity of
receptors allows the cell to
control the uptake of
particular ligands and the
distribution of these
ligands in the cell. The
early endosome serves as a
sorting compartment. The
illustration shows the 3
fates of the endocytosed
receptors and their ligands.
pH 6 induces dissociation
Multivesicular body
Late endosome
Endocytosed molecules that are
destined for the lysosome go from
the early endosome to the
multivesicular body to the late
endosome. Fusion of transport
vesicles carrying acid hydrolases
from the Golgi causes the late
endosome to mature into a lysosome.
In some cases, both the
receptor and the ligand
are transported to the
lysosome. This is the
case for EGF and its
receptor. EGF triggers a
cell to proliferate but the
signal is only required for
a short time. To limit the
response time both the
receptor and the ligand
are removed from the
membrane.
Transcytosis provides a way to deliver proteins
across an epithelium.
Transport of antibodies
in milk across the gut
epithelium of baby rats.
Acidic pH of the gut
favor association of
antibody with Fc
receptor whereas the
neutral pH of the
extracellular fluid
favors dissociation.
Exocytosis
Secretory vesicles
concentrate and store
products. Secreted
products can be either
small molecules or proteins.
Proteins originate at the
ER. In the Golgi, these
proteins aggregate and are
packaged into transport
vesicles as aggregates.
Insulin is a good
example of a
protein that is
stored in
secretory vesicles
until a cell
receives an signal
to secrete the
insulin.
Processing to the final
form occurs in the
secretory vesicle.
Removal of the
Pre-sequence (not
shown), folding
and disulfide bond
formation occur in
ER.
This is an example of a
protein that you would not
want to treat with
mercaptoethanol because
reduction of disulfide bonds
would inactivate the protein.
“pre-pro-proteins”
Some proteins are processed in secretory vesicles into multiple small
polypeptides. One explanation for this approach is that the small
polypeptides are too short to be cotranslationally transported into the
ER.
The End