50% of the cell volume is in membrane bound organelles

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(No Transcript)
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50 of the cell volume is in membrane bound
organelles
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Each organelle performs specific functions
Organelle Main function Structure
Organisms Chloroplast photosynthesis
double-memb. Plants, protists ER
modification, folding single-memb. All
eukaryotes of new proteins, lipids Golgi
sorting, modification single-memb.
All eukaryotes of new proteins Mito
energy production double-memb. Most
eukaryotes Vacuole storage housecleaning
single-memb. All eukaryotes Nucleus DNA
maintenance double-memb. All
eukaryotes RNA transcription
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General ruleOrganelles are generated from the
same type of organelle
nucleus
Nucleus (1)
mitochondria
Mitochondria (many/ribbon)
chloroplasts
Chloroplasts (many)
endoplasmic reticulum (ER)
endoplasmic reticulum (ER) (1)
growth
division
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Consequence during cell division, each daughter
cell needs at least one copy of each organelle
Different strategies
Nucleus very precise division and distribution
Mitochondria sufficient numbers, random
distribution
ER network is torn apart (perhaps also
vesiculation)
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But, not so clear for Golgi could be generated
from ER
For Golgi two different ideas
1. Golgi fragments during mitosis, then reforms
from fragments
2. Golgi is absorbed into ER during mitosis, then
reforms
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During zygote formation from gamete cells Egg
brings cytosol and all organelles Sperm brings
only nucleus All cytoplasmic organelles are
derived from mother! (only easily seen with DNA
containing organelles, such as mitochondria)
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Organelles have characteristic shapes
Some organelles are more or less spheres e.g.
peroxisomes, endosomes, lysosomes, secretory
granules Some organelles consist of sheets e.g.
Golgi stacks, inner and outer nuclear
membrane, outer mitochondrial membrane Some
organelles consist of tubules particularly the ER
network, mitochondrial inner membrane
(cristae), regions of Golgi How is the shape of
an organelle brought about and maintained?
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Membrane structure lipid bilayer and membrane
proteins
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Each organelle is characterized by a specific set
of proteins General rule every protein is found
at only one location (except when en route to its
final destination) Most organelles have luminal
and membrane proteins (exception Golgi has no
luminal proteins!)
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Lipids are also localized (although usually not
in an absolute manner) Cholesterol high in
plasma membrane, low in the ER (although it
is synthesized in the ER!) Phospho- inositides
different forms in different organelles (e.g.
PI3P in endosomes, PI4,5P2 in plasma membrane,
PI3,5P2 in lysosomes) Cardiolipin mitochondri
a
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How does each organelle receive its specific set
of lipids and proteins?Lipid transportProtein
transport
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Lipid transport is still mysterious
Questions How is cholesterol transported?
Probably not in vesicles How do
mitochondria receive their lipids? They must be
received from the ER, but how? How are lipids
transported from the outer to the inner
mitochondrial membrane? There is no continuity
between the two membranes How are lipids
flipped from the cytoplasmic leaflet of the ER
to the other leaflet of the bilayer?
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The ER is close to all other organelles in the
cell (mitochondria, lysosome, plasma membrane,
etc)
Could ABC transporter pump lipids from the ER to
other membranes?
ER
Membrane of other organelle
ABC (ATP-binding casette) transporter
ATPase domain
Phospholipid molecules
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Transport of cholesterol coupled to that of
phosphoinositol phosphate (Will Prinz, NIH)
Plasma membrane
ER membrane
phosphatase
PI4,5P2
cholesterol
Oxysterol binding protein (Osh4p)
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Protein transportSignals required to direct
proteins from the common site of synthesis in the
cytosol to their different destinationslike Zip
code system
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NO SIGNAL SEQUENCE
Cytoplasmic Nuclear Mitochondrial Plasma
membrane Secreted Resident ER and Golgi
Endosomes, Lysosomes
NUCLEAR LOCALIZATION SEQUENCE
MITOCHONDRIAL SIGNAL SEQUENCE
ER SIGNAL SEQUENCE
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The secretory pathway
Trans-Golgi Network (TGN)
medial
ribosome
cis
trans
ER
Golgi
Plasma membrane
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Experiments that led to the concept Pulse-chase
experiments (G. Palade)
Temperature sensitive VSVG mutant begins to
traffic at permissive temp.
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Two different phases of transport 1.
Translocation (in ER)
2. Vesicular transport
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• Signals must exist to direct proteins
• Signal sequences for translocation
• 2. Sorting or retention signals

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Translocation across the ER membrane
Soluble protein
Membrane protein
trans-membrane (TM) segment
Signal sequence
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SRP-dependent protein targeting to the ER
membrane (in mammals)
mRNA
ribosome
SRP (signal recognition particle)
signal sequence of emerging protein
translocon
SRP receptor
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Discovery of SRP (Walter Blobel, 1980)
Purified microsomal/rough ER membranes (from dog
pancreas)
microsomes
mRNA

translocation
Signal sequence
High salt wash
mRNA

no translocation
Signal sequence
Hydrophobic chromatography
translocation
SRP
Salt wash
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SRP contains 6 polypeptides
11S (250kDa)
SRP
SRP 54,68,72
Sucrose gradient
SRP 9,14,19
active fraction 11S
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SRP contains an essential RNA component, 7SL
(Walter Blobel 1982)
Noticed that UV absorbance of SRP at 260 nm gtgt
280 nm
Could it contain nucleic acid????
260nm absorbance could be reduced by acid
hydrolysis-----RNA
Is SRP activity sensitive to micrococcal
nuclease---------Yes
extracted RNA and ran on polyacrylamide gel-----
260 nts
Enzymatic cleavage reaction yield 40 nucleotides
at 3end
7SL
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Signal Recognition Particle (SRP) contains (6)
polypeptides, 1 7SL RNA
SRP RNA (7SL RNA)
This secondary structure is highly conserved
throughout eukaryotes!
Zweib et al.
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SRP-dependent protein targeting to the ER
membrane (in mammals)
mRNA
ribosome
SRP (signal recognition particle)
signal sequence of emerging protein
translocon
SRP receptor
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Identification of the SRP receptor in the ER
membrane
(Munro, Walter Blobel, 1981)
SRP
Salt washed microsomes
beads
1 detergent solubilize
beads
SRP receptor
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Membrane proteins have different topologies in ER
membrane
C
C
N
C
N
cytosol






ER
_
_
_
_


lumen
N
N
cleaved signal sequence
C
Type I
Type II
Type III
-reverse signal anchor
-signal anchor seq. (18-25 apolar) -not
cleaved -become ancored in membrane and
cause translocation of C-term
-cleavable signal seq. (7-15 apolar
residues) -SRP dependent -transmembrane
anchor stops translocation
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If only signal sequence translocation and then
secretion (soluble cargo) or plasma membrane
(membrane protein) (called default
pathway) Note If protein misfolded in ER- stuck
in ER)
Sorting and retention signals Retention in
organelle of secretory pathway, e.g. in ER This
is often a combination of retention and retrieval
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Retrieval signals for ER proteins Luminal
proteins
KDEL
Membrane proteins
KKXX
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Identification of KDEL sequence (ER lumen target
sequence) (Munro Pelham,
1987)
Some soluble proteins reside in the ER lumen- do
they have a sequence in common?
looked at sequence of (3) lumenal proteins
grp78, grp94, PDI
All (3) have KDEL at their C-terminus
Transfect COS cells with grp78, grp94, PDI (/-
KDEL) on each
35S radiolabel protein
Immunoprecipitated proteins from cells vs.
culture medium
Secreted or not?
(Lysozyme control KDEL)
sequence is also sufficient for retention
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Retention/retrieval of ER proteins
KDEL Retention/retrieval sequence
Golgi
low pH
KDEL receptor
Transport vesicles
ER
neutral pH
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If only signal sequence and no ER retention
sequence translocation and then secretion
(soluble cargo) or plasma membrane (membrane
protein) (called default pathway) Note If
protein misfolded in ER- stuck in ER)
Not entirely true, growing list of ER exit COPII
packaging signals
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The secretory pathway
Golgi
ER
Trans-Golgi Network (TGN)
medial
ribosome
cis
trans
C1
Plasma membrane
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Budding from ER in COPII coated vesicles
Golgi
ER
Trans-Golgi Network (TGN)
medial
ribosome
cis
trans
COPII vesicles
C1
Mogelsvang et al.
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COPII
Sec13
Sec23
Sec31
Sec24
Sar1
60-80nm COPII vesicles
COPII coat important for trafficking from ER
to Golgi helps shape membrane (stabilizes
curvature) Binds cargo via
Sec24 Associates with switch (Sar1/GTPase) so
that assembly is reversible
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COPII
Sec13
Sec23
Sec31
Sec24
Sar1
Sec24 binds cargo destined for Golgi- how does it
recognize diverse cargoes???
There is no universal ER export signal!
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(E. Miller Schekman et al. Cell, 2004)
Multiple regions have been found in various
proteins required for their export
Do all signals bind to the same site of Sec24?
Found mutant that no longer binds diacidic motif
of Sys1p, Bet1p---- does this mutant still bind
other trafficked substrates?
VSV-G, Sys1p, Bet1, diacidic motif does not
bind mutant Gap1p, Hip1p, Can1p diacidic
motif Does Emp24p, Erp1p, Erp2p,
ERGIC53 di-hydrophobic motif does not bind
mutant ERV41/ERV46 di-hydrophobic
motif Does Emp47p tyrosine motif does not
bind mutant Sed5 bipartite sorting signal does
not bind mutant Bos1p, Sec22p, unknown
sequence does not bind mutant Prm8p diphenylal
anine motif Does
Are their multiple binding sites on Sec24?
Sys1p, Bet1p bind to different region of Sec24
than Prm8p and Sec22p
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How does the COPII complex promote vesicle
budding?
COPII
Sec13
Sec23
Sec24
Sec31
Sar1
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Acceptor compartment
Donor compartment
Vesicles have a size of 60-90nm
Questions 1. How does a vesicle bud? How is
cargo concentrated? 2. How is a vesicle
targeted? 3. How does a vesicle fuse with its
target membrane?
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Vesicle budding requires coat proteins and the
generation of transient membrane curvature
COPII coated vesicle
ER exit sites Budding vesicles
Sar1p (GTP)
CopII
High curvature
Initial curvature
Sar1p (GTP)
Amphipathic helix
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Model for how COPII generates ER derived vesicles
Sec23/Sec24 Binds Sar1 and selects
cargo molecules
Sar1-GTP
Sec23
Sar1-GTP initiates coat formation
ER lumen
Vesicle (60nm)
Sec24
Sec13/31 Induces coat
polymerizaton and membrane deformation
ER lumen
(Bi/Goldberg et al.)
(Lee/Schekman et al.)
ER lumen
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How do proteins travel through subsequent Golgi
cisternae in stack
Golgi
Trans-Golgi Network (TGN)
medial
cis
trans
ER
C1
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Transport through the Golgi two models
1. Vesicular transport
Cargo moves forward in vesicles Golgi enzymes are
stationary
2. Cisternal maturation
Golgi enzymes move backwards in vesicles
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Cisternal maturation model
Stable cisternae model
Trans
cargo
Cis
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Evidence for Cisternal maturation model
(Losev/Glick Matsuura-Tokita/Nakano, Nature
2006)
Simple question previously limited by the
resolution of light microscope
Most eukaryotes
Saccharomyces Cerevisiae
trans
xxxxxxx
xxxxxxx
xxxxxxx
cis
So, can follow individual cisternae
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Evidence for Cisternal maturation model
(Losev/Glick Matsuura-Tokita/Nakano, Nature
2006)
SC yeast express GFP-Rer1 (cis Golgi, green)
and mRFP-Gos1 (trans Golgi, red)
Does a green cisternae turn red?? Or stay
green???
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Golgi Cisternal maturation model
Questions remain Can you visualize cargo
proteins simultaneously? What happens in cells
where the golgi is stacked?
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Why is the Golgi organized in stacks
Most eukaryotes
trans
xxxxxxx
Many secreted proteins are processed in Golgi by
subsequent steps, these steps are
spatially separated by separating the cisternae
in the golgi Like an assembly line
xxxxxxx
xxxxxxx
cis
Proteins that stack the golgi have not been
identified!
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Transport beyond the golgi to the plasma membrane
Golgi
ER
Trans-Golgi Network (TGN)
medial
ribosome
cis
trans
COPII
C1
COPI
Back to ER
Plasma membrane
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How does cargo find its target membrane to fuse
with?
Vesicles travel on Microtubules
ER to Golgi transport Towards the minus end of
microtubules
Golgi to PM transport Towards the plus end of
microtubules
Snare pairing drives fusion with target membrane
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Fusion of vesicles with target membrane requires
SNARE pairs
Target membrane
Vesicle
t-SNARE
heavy
v-SNARE
N
C
C
N
N
light
C
C
Golgi to PM v-SNARE Snc1 t-SNARE heavy
chain Sso1 light chains Sec9(N),
Sec9(C) ER to cis-Golgi v-SNARE
Bet1 t-SNARE heavy chain Sed5
light chains Bos1, Sec22
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Only certain combinations of SNAREs result in
fusion (synaptic vesicles)
Vesicle
v-SNARE (VAMP)
C
N
C
N
Syntaxin
C
SNAP25
C
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Sar1p N-Terminal Helix Initiates Membrane
Curvature and Completes the Fission of
a COPII vesicle (Lee et al. 2005, Cell)
Bliak Main question of paper Cope Figure
1 Haines Figure 2a, 2b Kennerly Figure 2c,
2d Patzlaff Figure 3 Rao Figure
4a Rebbapragada Figure 4b Rex Figure
4c Ross Figure 5a, 5b Sanchez Figure 5c,
5d Schaaf Figure 6a Spindler Figure 6b,
6c Stockburger Figure 6d Wang Figure
7 Zhang Figure 8 Zhou Conclusions Zurek What
did you like about this paper? Why do you think
this was a Cell paper?
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When presenting figures

• What is the question that the figure is asking
• What is the experiment- be detailed about how it
  is done
• What is the result
• Does it look pretty convincing

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Identification of the KDEL receptor (Hugh Pelham)

• Isolate mutants in yeast that fail to retain ER
  resident proteins
• ? Erd2p (multi-spanning membrane protein)
• Demonstration that this is the receptor
• S. cerevisiae recognizes HDEL
• (but not DDEL)
• K. lactis (a related yeast) recognizes
  bothHDEL
• and DDEL
• ? replace S. cerevisiae Erd2p with K. lactis
  Erd2p
• ? now DDEL recognized in S. cerevisiae