Transcript Lecture Slides 2

Chapter 12
• Intracellular
Compartments and
Protein Sorting
Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)
Protein Movement between Compartments
*
Most proteins are synthesized on cytoplasmic ribosomes and
must be delivered to their ultimate compartment of residence.
*
Proteins contain sorting signals that direct their movement
throughout the cell.
*
These sorting signals are recognized by specific receptors that
mediate delivery to the appropriate organelle.
*
There are three major types of protein traffic between
compartments:
1) Gated transport
2) Transmembrane translocation
3) Vesicular transport
Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)
Gated Transport
(Nuclear Import/Export)
Figure 12-10 Molecular Biology of the Cell (© Garland Science 2008)
Transmembrane Translocation
* Proteins are directly translocated across the membrane
bilayer.
*
Translocation is performed by a membrane protein
complex that forms a translocation pore.
*
Proteins pass through the membrane bilayer as unfolded
chains.
*
Two major strategies are used to accomplish this feat:
co-translational & post-translational import.
Vesicular
Transport
Figure 12-7
Protein sorting signals
Signal sequences direct protein delivery
SS
Destination
Mitochondria
Cytoplasm
1. Deletion of signal sequence (SS)
Cytoplasm
2. Addition of a signal sequence
Mitochondria
SS
Mitochondrial Protein Import
*
Mitochondria utilize the energy from electron transport and oxidative
phosphorylation to synthesize the majority of the cell's ATP.
*
Most mitochondrial proteins are synthesized on cytoplasmic ribosomes
and are post-translationally imported into this organelle.
*
Because of the double membrane surrounding this organelle, there are
four targets for mitochondrial proteins:
1. Outer membrane
3. Inner membrane
2. Intermembrane space
4. Matrix space
*
Mitochondrial proteins usually contain an N-terminal targeting sequence
that is capable of forming an amphipathic -helix; positively-charged
residues are clustered on one side of the helix and uncharged residues
are present on the other.
*
The mitochondrial outer membrane contains specific receptor proteins
that bind to the mitochondrial targeting signal.
The four compartments within mitochondria
Figure 12-21 Molecular Biology of the Cell (© Garland Science 2008)
* Signal sequence for
mitochondrial protein import.
*
Note the amphipathic nature
of the -helix.
Figure 12-22
Protein translocators
in mitochondrial
membranes
(Matrix/Inner Membrane)
(Inner Membrane)
Figure 12-23
Mitochondrial Protein Import (cont’d)
*
Translocation into the mitochondrial matrix requires both ATP
hydrolysis and an electrochemical gradient across the inner
mitochondrial membrane.
*
Translocation occurs at sites where the inner and outer
membrane are in close apposition. These regions are known
as contact sites.
*
Proteins are imported into the mitochondria in an unfolded
state.
*
Maintenance in an unfolded state is mediated by hsp70
proteins that act as molecular chaperones.
*
Protein transport into the inner membrane or intermembrane
space requires additional targeting signals.
*
Much of our current knowledge of mitochondrial protein import
has come from in vitro studies with isolated mitochondria.
Protein import into mitochondria
Figure 12-25 Molecular Biology of the Cell (© Garland Science 2008)
The role of energy in mitochondrial
protein import
Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008)
The hsp70 family of molecular chaperones
Figure 6-86 Molecular Biology of the Cell (© Garland Science 2008)
The role of energy in mitochondrial
protein import
Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-28 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-28b Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-28 Molecular Biology of the Cell (© Garland Science 2008)
Studying mitochondrial protein import in vitro
*
Isolated mitochondria are mixed with the radioactivelylabeled protein to be studied
?
IMPORT
?
IMPORT
*
Import may be detected by one of the following methods:
1) Density gradient centrifugation; if imported, proteins will
fractionate with the organelle.
2) SDS-PAGE analysis to determine if the signal sequence was
removed during the import reaction.
3) Protease protection assays; imported protein will be
protected from the action of added proteases.
*
By adding or removing different components from the import reaction,
one can determine the requirements for protein import.
in vitro studies of mitochondrial protein
import
Figure 12-24 Molecular Biology of the Cell (© Garland Science 2008)
Secretory
Pathway
Proteins enter into the secretory pathway at the ER where they are co-translationally
inserted into the ER membrane.
Proteins then travel to successive organelles via membrane-bound intermediates.
Endoplasmic Reticulum
Functions of the ER
*
The entry point for proteins that proceed through the
secretory pathway.
*
Modification of proteins: a predominant modification is the
glycosylation of specific asparagine residues (N-linked
sugars).
*
Quality control: proteins must be properly folded before
they are allowed to leave the ER. Proteins that fail to
achieve a native state are degraded.
* Sequestration of Ca++ from the cytoplasm.
*
Primary site of lipid biosynthesis.
Figure 12-35 Molecular Biology of the Cell (© Garland Science 2008)
Abundant smooth ER
in steroid-hormonesecreting cell
A 3-D reconstruction
of ER in liver cell
Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)
Rough ER in pancreatic
exocrine cell
Free
Membranebound
Figure 12-41a
Separating the Smooth & Rough ER
Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)
The Signal
Hypothesis
Figure 12-38
George Palade: 1974 Nobel Prize in Medicine
Protein Import into the ER
Step 1: Establishing a tight interaction with the ER membrane
*
A hydrophobic signal peptide, usually at the N-terminus of
the protein, directs entry into the ER.
*
The signal peptide is recognized by the Signal Recognition
Particle (SRP) as soon as it emerges from the ribosome.
This interaction arrests translation.
* The ER membrane contains an SRP receptor that mediates
the initial association of the SRP-ribosome complex with the
cytoplasmic face of the ER.
*
The ribosome subsequently associates with a translocation
complex (the Sec61 complex) in the ER membrane and the
SRP is released back into the cytosol.
Table 12-3 Molecular Biology of the Cell (© Garland Science 2008)
Signal-recognition
particle (SRP)
Figure 12-39a
Two functions for the signal sequence:
1. Targets protein/ribosome complex to the ER membrane.
2. Serves as a start-transfer sequence that opens translocation pore.
Figure 12-40 Molecular Biology of the Cell (© Garland Science 2008)
Protein Import into the ER
Step 2: Co-translational translocation of the polypeptide
*
Upon association with the ER, the ribosome resumes translation
and co-translationally inserts the polypeptide chain into the ER
lumen through a translocation pore.
*
The protein is passed through the membrane as a single,
unfolded chain and folds into its native conformation within the
ER lumen. This folding process requires protein chaperones.
*
The N-terminal signal peptide is removed by Signal Peptidase, a
protease present in the lumen of the ER.
*
Integral membrane proteins contain "stop transfer" sequences
that result in a block to the translocation process.
Structure of the Sec61 translocation complex
Figure 12-42 Molecular Biology of the Cell (© Garland Science 2008)
A ribosome
bound to the
Sec61 protein
translocator
Figure 12-43 Molecular Biology of the Cell (© Garland Science 2008)
Translocation of a soluble protein
Figure 12-45 Molecular Biology of the Cell (© Garland Science 2008)
A single-pass transmembrane protein
Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008)
Integration of a singlepass membrane protein
with an internal signal
sequence
NOTE: Orientation
across membrane bilayer.
Figure 12-47 Molecular Biology of the Cell (© Garland Science 2008)
A double-pass transmembrane protein
Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008)
Insertion of a multipass membrane protein into the
ER
Figure 12-49 Molecular Biology of the Cell (© Garland Science 2008)
Genetic approaches for studying the mechanism of protein
translocation
Wild-type
Engineered Cell
Enzyme in
cytosol: cell
lives without
histidine
Enzyme targeted
to ER: cell dies
without histidine
Mutant Engineered Cell
Not all enzyme
targeted to ER:
cell lives without
histidine
Panel 12-1
*
Most proteins in the secretory
pathway are glycosylated;
modified by the addition of sugar
residues.
*
A precursor oligosaccharide unit
is added to particular asparagine
residues (N-linked carbohydrate).
Figure 12-50 Molecular Biology of the Cell (© Garland Science 2008)
Protein glycosylation in the rough ER
Figure 12-51
Synthesis of the lipidlinked precursor
oligosaccharide in the
rough ER membrane
Figure 12-52
Possible functions for the N-linked
oligosaccharide chains?
1. Promoting protein folding & stability.
2. Protecting the protein from proteolysis.
3. Serving as a targeting determinant.
4. Facilitating or directing anterograde (forward)
transport.
5. Promoting cell-to-cell adhesion.
The role of N-linked glycosylation in ER protein folding
Figure 12-53
The export & degradation of misfolded ER proteins
* The ER functions as a quality
control organelle.
* Proteins that are not properly
folded are exported from the
ER and degraded in the
cytosol.
Figure 12-54
Quality control in the ER and Cystic Fibrosis
*
A particular deletion that removes three nucleotides in the Cftr gene is the
most common mutation responsible for this disease.
*
*
This deletion results in the removal of a phenylalanine residue, F508.
*
However, the encoded protein would be FUNCTIONAL if it was allowed to
go to the plasma membrane.
The encoded protein is recognized by the ER quality-control machinery and
is ultimately targeted for degradation (in the cytoplasm).
Plasma membrane
M
*
Degradation
Knowing the above, how
might you try to treat CF
patients that possess this
cftr allele?
Lipid synthesis in the ER
*
The cytoplasmic half of the ER bilayer is the primary site of
phospholipid synthesis. The enzymes that catalyze these
reactions are ER membrane proteins whose active sites
face the cytosol.
*
Phospholipid translocators function to "flip" specific
phospholipids from one half of the bilayer to the other.
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Specific phospholipid transfer proteins (PLTPs) transport
phospholipids from the ER to mitochondria and
peroxisomes.
Synthesis of phosphatidylcholine
Figure 12-57 Molecular Biology of the Cell (© Garland Science 2008)
The role of phospholipid translocators in lipid bilayer synthesis
SCRAMBLASE
FLIPPASE
Figure 12-58
Phospholipid exchange/transfer proteins
Ch. 13:
Intracellular Vesicular Traffic
Figure 13-2 Molecular Biology of the Cell (© Garland Science 2008)
Biosynthetic-Secretory/Endocytic Pathway
Figure 13-3 Molecular Biology of the Cell (© Garland Science 2008)
Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008)
Vesicular Transport
*
The lumen of each compartment communicating by way
of vesicular traffic is topologically equivalent.
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The two primary pathways for vesicular traffic are
known as the biosynthetic-secretory and the endocytic
pathways.
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A transport vesicle must select the cargo to be
transported to the next compartment and exclude that
which is to remain behind.
*
To ensure compartment identity, a transport vesicle
must fuse only with the appropriate target organelle.
Protein coats facilitate multiple steps of vesicular transport
Coated Vesicles
*
Most transport vesicles form from specialized "coated" regions of
the membrane and bud off as coated vesicles.
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These coats are protein structures that form on the cytosolic face
of a membrane region that will form the transport vesicle.
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Several coat structures have been identified in eukaryotic cells
and each appears to perform a distinct transport function.
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Coated vesicles generally mediate the directional flow of specific
types of membranes.
*
The assembly of a coat structure on a membrane may be the
driving force in bud formation.
*
Coat proteins play an important role in the selection of vesicle
cargo.
Three examples of coated vesicles
Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)
Generation of
membrane curvature
a. Membrane deformation by proteins that exert
mechanical force.
b. Curvature generation by scaffolding proteins
(coat proteins).
c. Curvature generation by a hydrophobic
insertion (wedging) mechanism.
Different coated vesicles mediate distinct
transport steps within the secretory pathway
Figure 13-5 Molecular Biology of the Cell (© Garland Science 2008)
Clathrin-coated Vesicles
*
The primary component of one membrane coat is
clathrin, a large protein complex composed of three
subunits each of a heavy chain and a light chain.
*
Clathrin-coated vesicles mediate Golgi-to-lysosome
protein delivery and plasma membrane receptormediated endocytosis.
*
Clathrin coat assembly provides the mechanical force
necessary for bud emergence and vesicle formation.
* Other proteins in this coat, known as adaptins, mediate
the binding and sequestration of specific transmembrane
receptors and their bound cargo.
Clathrin-coated pits
& vesicles on the
inner surface of the
p.m. in cultured
fibroblasts
Figure 13-6 Molecular Biology of the Cell (© Garland Science 2008)
The structure
of a clathrin coat
2
3
Clathrin light
chain
Assembled
clathrin
6
4
Clathrin heavy
chain
Clathrin
triskelia
5
Figure 13-7
Clathrin coat assembly & disassembly
Figure 13-8 Molecular Biology of the Cell (© Garland Science 2008)
Ligand
cargo
2
1
Receptor
cargo
Nucleation
determinant
3
Clathrin light
chain
Adaptor
Assembled
clathrin
6
4
Clathrin heavy
chain
Clathrin
triskelia
7
5
Uncoated vesicle
Assembled clathrin at the
plasma membrane
Clathrin basket
Figure 2
Receptor-mediated endocytosis and clathrin morphology. T he numbered sequence of events during receptor-mediated endocytosis is
shown clockwise from upper left. ,
Adaptors (AP2) are recruited by a nucleation determinant, involving PtdIns(4,5)P 2 , in a
process that requires cooperative interplay with clathrin and cargo (orange triangles and purple circles).
Clathrin triskelia from the
cytosol coassemble with adaptors and cargo, causing cargo sequestration and sorting. T rimerized clathrin heavy chains (red ) are based
Cargo selection by the clathrin coat
The role of dynamin in
pinching off clathrincoated vesicles from
the membrane.
Figure 13-12 Molecular Biology of the Cell (© Garland Science 2008)
Monomeric GTPases control coat assembly
Figure 13-13a
“Inactive”
(GDP)
GTP-binding proteins
act as molecular
switches
(GTP)
GEF = Guanine nucleotide
Exchange Factor
“Active”
Figure 3-73
Molecular Biology of the Cell
GAP = GTPase-Activating
Protein
Monomeric GTPases control coat assembly
Sar1- COP II
ARF - COP I
Clathrin
Figure 13-13a
Coat recruitment & cargo selection
Figure 13-13b Molecular Biology of the Cell (© Garland Science 2008)
Formation of COPII-coated vesicles
Sec13/31 “cage”
Figure 13-13
How does a
transport vesicle
find its correct
destination?
Vesicle Targeting
*
To ensure compartment identity, transport vesicles must fuse
only with the appropriate target membrane.
*
This specificity is mediated by two classes of proteins: the
Rab family of monomeric GTPases and the transmembrane
SNARE proteins that mediate membrane fusion.
*
The active GTP-bound Rab proteins interact with a diverse
set of Ras effector proteins that mediate vesicle transport,
tethering and fusion to the appropriate target membrane.
*
Membrane fusion is facilitated by the pairing of
complementary transmembrane receptors present on the
vesicle (v-SNAREs) and the target membrane (t-SNAREs).
*
SNARE complex disassembly after membrane fusion is
catalyzed by NSF, a cytoplasmic ATPase.
Tethering of a vesicle to a
target membrane
Figure 13-14 Molecular Biology of the Cell (© Garland Science 2008)
The structure of a trans-SNARE complex
Figure 13-16 Molecular Biology of the Cell (© Garland Science 2008)
A model for how
SNARE proteins may
catalyze membrane
fusion
Figure 13-17 Molecular Biology of the Cell (© Garland Science 2008)
NSF facilitates the dissociation of SNARE proteins
after membrane fusion
Figure 13-18 Molecular Biology of the Cell (© Garland Science 2008)