The Secretory Pathway

Report
GBS709: Biological Organization- Secretion
David M. Bedwell, Ph.D.
Office: BBRB432
Phone: 934-6593
E-mail: [email protected]
Reading: Alberts et al., Molecular Biology of the Cell (5th Ed.):
Endoplasmic Reticulum: Chapter 12, pp. 723-745
Vesicular Traffic:
Chapter 13, pp. 749-787
Major Intracellular Compartments of an Animal Cell
Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)
Topological Relationships Between
Compartments of the Secretory Pathway
Figure 12-5 Molecular Biology of the Cell (© Garland Science 2008)
Simplified Roadmap of Intracellular Protein Traffic
Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)
Table 12-3 Molecular Biology of the Cell (© Garland Science 2008)
General Features of the ER
 The endoplasmic reticulum (ER) is a highly convoluted, single
membrane-bound organelle that contains as much as 50% of
the total membrane of liver cells.
 Due to tubular nature of the ER, the total membrane surface
area of ER in liver cells is ~25 times the surface area of the
plasma membrane.
 The ER membrane is thought to enclose a single internal space
called the lumen, which can comprise >10% of the total cell
volume.
The ER Frequently Exhibits a Reticular Structure
Fluorescent visualization of the endoplasmic reticulum (ER) showing
it frequently exists as a tubular network throughout the cytosol.
The Reticular Portion of the ER Is a
Highly Dynamic Tubular Network
Rough and Smooth ER
 The smooth ER (SER) is the site of most lipid biosynthesis in
the cell.
 The rough ER (RER) is the site of entry of proteins into the
secretory pathway, and is so named because ribosomes
located on the rough ER (carrying out the translocation of
proteins into the ER) give this membrane a "rough" appearance
when viewed under the microscope.
 Transitional elements (part rough and part smooth) are the site
at which transport vesicles bud off to carry newly synthesized
lipids and proteins to the Golgi apparatus.
Rough and Smooth ER
Rough and smooth ER in a liver cell
Figure 12-36 Molecular Biology of the Cell (© Garland Science 2008)
Isolation of Rough and Smooth
Microsomes from the ER
Figure 12-37 Molecular Biology of the Cell (© Garland Science 2008)
The Signal Hypothesis
[Blobel and Dobberstein, J. Cell Biol. 67: 835 (1975)]
Gunter Blobel won the 1999
Nobel Prize for discovering
the mechanism of ER
protein targeting.
Figure 12-38 Molecular Biology of the Cell (© Garland Science 2008)
Evidence for a Proteinaceous Channel
 Rough microsomes contain channels of high ion conductivity
that can be activated by puromycin, an antibiotic that causes the
release of nascent chains from ribosomes.
 This suggested that when the nascent chain is released, it
leaves a "hole" (the postulated protein conducting channel) in
the ER membrane.
 This channel remained open until the membranes were washed
with high salt, which caused the ribosomes to dissociate into
their subunits.
 These results are most consistent with a model in which a
protein-lined channel in the ER membrane opens when a
ribosome binds to the membrane and resumes translocation.
 When the ribosome is tricked into releasing the nascent chain
prematurely with puromycin, the channel remains open (thus
allowing ions to flow through) until the ribosome dissociates,
which causes the channel to close.
Overview of ER Translocation
The translocation of proteins into the ER is generally a cotranslational process and can be divided into two phases:
 targeting of the nascent polypeptide to the cytoplasmic
surface of the ER membrane.
 translocation of the polypeptide across the ER
membrane.
Targeting Secretory Proteins to the ER
 In eukaryotic cells, many proteins are targeted to the ER by a
ribonucleoprotein complex called the Signal Recognition Particle
(SRP). Targeting occurs in a co-translational manner.
 Once ~30 amino acids of the nascent chain emerge from the
ribosome, it is bound by the SRP and translation is temporarily
arrested (this represents ~ 70 amino acids total, since 30-40
amino acids are buried within the ribosome).
 Following the translational arrest, the SRP-ribosome complex
moves to the ER membrane where SRP docks to a specific
protein complex on the ER surface called the SRP receptor (the
ribosome may also associate with a ribosome receptor on the
ER membrane).
 The translational arrest is relieved when the SRP releases and
recycles for another round, allowing translation of the ER bound
secretory protein to resume.
Signal Recognition Particle (SRP)
(Binds SRP Receptor)
SRP is an 11S ribonucleoprotein
(RNP) complex composed of a 7S
RNA (~300 nucleotides) and six
polypeptide chains.
Signal Recognition Particle (SRP)
The SRP contains three functional domains:
 The p19/54 domain binds to the signal sequence. p54 binds the
signal sequence, while p19 links p54 to the 7S RNA.
 The p9/14 domain binds to the A site of the ribosome and arrests
translation elongation.
 The p68/72 domain helps initiate insertion of the signal sequence
into the ER membrane by binding to the SRP receptor.
Signal Recognition Particle (SRP)
Mammalian SRP
Figure 12-39 Molecular Biology of the Cell (© Garland Science 2008)
SRP bound to a ribosome
How an ER Signal and SRP
Direct Ribosomes to the ER
Figure 12-40 Molecular Biology of the Cell (© Garland Science 2008)
Pools of Free and Membrane Bound Ribosomes
Figure 12-41 Molecular Biology of the Cell (© Garland Science 2008)
Structure of the Sec61 Complex
Figure 12-42 Molecular Biology of the Cell (© Garland Science 2008)
Ribosome Bound to the
Eukaryotic ER Protein Translocator
Figure 12-43 Molecular Biology of the Cell (© Garland Science 2008)
Cryo-EM Reconstruction Movie of the
Yeast Ribosome and ER Translocator
Components of the ER Translocase- Sec61p
 Sec61p has been found to interact directly with translocating
polypeptides by crosslinking experiments in yeast and
mammalian cells (the yeast and mammalian forms of Sec61p
are 56% identical). Sec61p also interacts directly with
ribosomes, and also functions as a ribosome receptor.
 Crosslinking of Sec61p to translocating precursors occurs in the
ATP-dependent phase of the transport reaction, indicating that
the interaction occurs while the precursor is moving across the
membrane.
 Interestingly, Sec61p is a polytopic membrane protein that
contains 10 membrane spanning domains, and shares structural
and amino acid sequence homology with the E. coli SecY
protein.
Components of the ER Translocase- BiP
 BiP stands for Immunoglobulin Heavy Chain Binding Protein,
and is called Kar2p in yeast. BiP is a resident ER protein that
is related to the Hsp70 class of proteins and is thought to bind
aggregated or misfolded proteins to prevent them from leaving
the ER. It may also help refold them using an ATP dependent
activity.
 BiP can also be crosslinked to translocating precursors in
yeast. This result is also consistent with studies where it was
found that mutationally altered Kar2p blocked not only
precursor translocation, but also precursor interaction with
Sec61p
Peripheral Components of the ER Translocase
 Other proteins (Sec62, Sec63, Sec71 and Sec72) can assist with
post-translational import. Sec63 contains a J domain and binds BiP.
 Signal peptidase- The signal peptidase has been purified as a
complex from both mammalian and yeast microsomes, and each
enzyme contains five different proteins. The catalytic site is on the
trans (lumenal) side of the ER membrane. The subunits of this
complex appear to span the membrane only once and have a
second hydrophobic region that may interact with signal sequences.
 Oligosaccharyltransferase is the enzyme responsible for the transfer
of an oligosaccharyl moiety to an asparagine residue in a consensus
(Asn-X-Ser/Thr) recognition motif. The mammalian enzyme consists
of three subunits.
 Neither signal peptidase or the oligosaccharyltransferase are
required for protein translocation.
3 Ways for Protein Translocation Through
Structurally Similar Translocators
Figure 12-44 Molecular Biology of the Cell (© Garland Science 2008)
Yeast Genetic Analysis Played a Key Role in
Identifying Key Proteins in the Secretory Pathway
Membrane Protein Insertion: Model For Soluble
Protein Translocation Across the ER Membrane
Figure 12-45 Molecular Biology of the Cell (© Garland Science 2008)
Membrane Protein Insertion: Model For
Integration of a Single-Pass Transmembrane
Protein into the ER Membrane
Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008)
Membrane Protein Insertion: Integration of SinglePass Transmembrane Proteins with an Internal
Signal into the ER Membrane
Figure 12-47 Molecular Biology of the Cell (© Garland Science 2008)
Membrane Protein Insertion: Integration of a DoublePass Transmembrane Protein with an Internal Signal
into the ER Membrane
Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008)
Membrane Protein Insertion: Insertion of the Multipass
Membrane Protein Rhodopsin into the ER Membrane
Figure 12-49 Molecular Biology of the Cell (© Garland Science 2008)
Asparagine-Linked (N-Linked) Precursor
Oligosaccharides are Added to Proteins in the ER
Figure 12-50 Molecular Biology of the Cell (© Garland Science 2008)
Protein Glycosylation in the Rough ER
Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008)
N-Linked Glycosylation Helps
the ER Monitor Protein Folding
Figure 12-53 Molecular Biology of the Cell (© Garland Science 2008)
Export and Degradation of Misfolded ER Proteins via
ER-Associated Decay (ERAD)
Figure 12-54 Molecular Biology of the Cell (© Garland Science 2008)
The Unfolded Protein Response (UPR)
 The unfolded protein response (UPR) results from the
accumulation of misfolded proteins in the ER due to cellular
or environmental stress.
 UPR triggers activation of a wide range of genes encoding
proteins involved in protein folding and glycosylation in the
ER, proteins involved in ER Associated Decay (ERAD), and
proteins involved in vesicular transport.
 Thus, quality control, ERAD and UPR are tightly coordinated
processes.
3 Parallel Signaling Pathways Activated by the
Unfolded Protein Response (UPR)
Figure 12-55a Molecular Biology of the Cell (© Garland Science 2008)
Comparison of Mammalian and Yeast UPR
 Basic features of mammalian and yeast UPR are similar, but
mammalian UPR has more “bells & whistles”.
- Mammals & yeast: Ire1p sensor/kinase serves to splice the XBP1
mRNA, allowing the translation of the transcription factor Xbp1.
- Mammals only: Another transcription factor, ATF6, is cleaved
directly from the cytoplasmic domain of an ER transmembrane
sensor.
- Mammals only: Translational repression is mediated by another
transmembrane sensor/kinase, PERK.
 Together, these responses to unfolded proteins in the ER allows
mammalian cell to coordinately regulate transcription and translation
to rapidly recover from ER stress. If the stress is too severe,
apoptosis is induced.
Transmembrane ER Stress Sensors
That Regulate the UPR
IRE1 splices
XBP1 mRNA
PERK blocks
translation
initiation
ATF6 contains
a transcription
factor
Regulated XBP1 mRNA Splicing is One Pathway
Activated During the Unfolded Protein Response
Ire1p
XBP1
Xbp1
Figure 12-55b Molecular Biology of the Cell (© Garland Science 2008)
Induction of Transcription During the UPR
by XBP1 and ATF6
Regulation of Translation During the UPR
 eIF2 phosphorylation
inhibits bulk translation.
 The presence of uORFs
in certain mRNAs allows
the translation of specific
proteins during the UPR
that turn on UPRresponsive genes.
ER Questions?
Proteins Transit From the ER to the Golgi
Apparatus and Through the Secretory Pathway
Occurs via Vesicular Transport
Figure 13-3 Molecular Biology of the Cell (© Garland Science 2008)
Basic Features of Vesicular Transport
Figure 13-2 Molecular Biology of the Cell (© Garland Science 2008)
3 types of Vesicle Coat Proteins
Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)
Use of Different Coats in Vesicular Traffic
Figure 13-5 Molecular Biology of the Cell (© Garland Science 2008)
Different Phosphoinositides Mark Organelles
and Membrane Domains
Phosphotidylinositol (PI) and
Phosphoinositides (PIPs)
Figure 13-10 Molecular Biology of the Cell (© Garland Science 2008)
Localized kinases and phosphatases
in specific membranes regulate the
production of PIPs which recruit binding
proteins to specific membranes
Intracellular Location of Phosphoinositides
Figure 13-11 Molecular Biology of the Cell (© Garland Science 2008)
Monomeric GTPases That Control Coat Assembly
Monomeric GTPases Control Coat Assembly
 Arf proteins mediate COPI and clathrin coat assembly at Golgi
membranes.
 Sar1 protein mediates COPII coat assembly at the ER membrane.
Rab proteins guide vesicle targeting
 >60 members in Rab family. Each binds to specific membranes.
 Each Rab is associated with one or more membrane-enclosed
organelles secretory or endocytic pathways.
 Each organelle has at least one Rab on its cytosolic surface.
 Soluble Rab-GDI (GDP dissociation inhibitor) keeps Rabs soluble
and inactive.
 Membrane bound Rab-GEF (GTP Exchange Factor) activate Rabs
via GTP-binding.
 Rab-GTP binds Rab effectors on membranes that mediate
vesicular transport, membrane tethering and fusion.
Formation of COPII-Coated Vesicles
at the ER Membrane
- Sar1 is a coat-recruitment GTPase.
- Sar1-GEF is a GDP/GTP exchange factor.
- Activated Sar1-GTP binds membrane
via an amphipathic helix.
- Sar1 binding initiates membrane curvature.
Figure 13-13a/b Molecular Biology of the Cell (© Garland Science 2008)
- Sar1 recruits a complex of COPII coat
proteins (Sec23/24).
- Sec24 binds cytoplasmic tails of cargo
receptors.
- COPII coat curves membrane of vesicle.
Formation of COPII-Coated Vesicles
at the ER Membrane
- 2 additional COPII coat proteins, Sec13/31,
forms the outer sell of the coat.
- Sec13/31 can assemble into cages with the
dimensions of COPII-coated vesicles.
Figure 13-13c/d Molecular Biology of the Cell (© Garland Science 2008)
- Membrane-bound, active Sar1-GTP recruits
COPII subunits to the membrane.
- This causes a membrane bud to form, which
includes selected transmembrane proteins
as cargo.
- A subsequent membrane fusion event
pinches off the coated vesicle.
Tethering of a Vesicle to a Target Membrane
Figure 13-14 Molecular Biology of the Cell (© Garland Science 2008)
Structure of a Trans-SNARE Complex
Figure 13-16 Molecular Biology of the Cell (© Garland Science 2008)
Model of How SNARE Proteins Catalyze
Membrane Fusion
Figure 13-17 Molecular Biology of the Cell (© Garland Science 2008)
NEM-Sensitive Factor (NSF) Dissociates
SNARE Pairs After a Membrane Fusion Cycle
N-Ethylmaleimide (NEM) is a thiol-reactive compound that inactivates NSF.
Figure 13-18 Molecular Biology of the Cell (© Garland Science 2008)
Recruitment of Cargo Molecules
into ER Transport Vesicles
A typical transport vesicle
contains about 200 membrane
proteins as cargo.
Figure 13-20 Molecular Biology of the Cell (© Garland Science 2008)
Retention of Incompletely Assembled
Antibody Molecules in the ER
Figure 13-21 Molecular Biology of the Cell (© Garland Science 2008)
Homotypic Membrane Fusion
Figure 13-22 Molecular Biology of the Cell (© Garland Science 2008)
Vesicular Tubular Clusters Form Between
the ER and the cis Golgi Network
Figure 13-23a/b Molecular Biology of the Cell (© Garland Science 2008)
Model for the Retrieval of
Soluble ER Resident Proteins
Figure 13-24a Molecular Biology of the Cell (© Garland Science 2008)
The Golgi Apparatus
Figure 13-25a/b Molecular Biology of the Cell (© Garland Science 2008)
Oligosaccharide Processing
in Golgi Compartments
Figure 13-28 Molecular Biology of the Cell (© Garland Science 2008)
2 Classes of N-Linked Oligosaccharides
Found in Mature Mammalian Glycoproteins
Figure 13-30 Molecular Biology of the Cell (© Garland Science 2008)
Oligosaccharide Processing in the ER
and Golgi Apparatus
Figure 13-31 Molecular Biology of the Cell (© Garland Science 2008)
N- and O-Linked Glycosylation
Figure 13-32 Molecular Biology of the Cell (© Garland Science 2008)
What’s the Purpose of Glycosylation?
 Makes protein folding intermediates more soluble and prevents
protein aggregation.
 Sequential modifications of N-linked sugars make a “glyco-code”
that marks the progression of protein folding and mediates the
binding of protein to chaperones and lectins (e.g., in ER quality
control).
 Sugars have limited flexibility. This can limit the approach of
other proteins (such as proteases) to the glycoprotein surface
and reduce proteolysis.
 Extensive glycosylation of proteins (e.g., in the mucus coat of
lungs and intestinal cells) protects against many pathogens.
 Recognition of glycoprotein sugar chains by lectins in the
extracellular space is important for developmental processes
and cell-cell recognition.
2 Models to Explain Golgi Organization and
Protein Transport Between Cisternae
Both mechanisms are probably used to
maintain Golgi compartmental integrity
Figure 13-35 Molecular Biology of the Cell (© Garland Science 2008)
Golgi Questions?

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