Section 3

Cytoplasmic
Membrane Systems
Chapter 8
Cytoplasmic Membrane
Systems: Structure, Function,
and Membrane Trafcking
Required Reading
pages 270-305, 308-317
( S e c . 8 . 1 - 8 . 6 , 8 . 8 , 8 . 9 )
P L U S
C h a p t e r 1 2 , p a g e s 5 4 1 - 5 4 2
( S e c . 1 2 . 7 )
Clicker Quiz #4
Will be on...
From the beginning of Section 3
to (but not including) the slide
that says Membrane Lipid Synthesis
1
2
3
Eukaryotic Cell Internal Structure
Goal of this section
To answer how:
1. The cell synthesizes components of the membranes
2. The cell ensures that the correct molecules arrives at
the correct membrane, or inside the appropriate organelle

Compartments are not

independent - they form
an integrated
functional unit
4
5
6
- all membranes are constantly in flux
- very dynamic
- compartments all depend on one another
- compartments all work together
- membranes take on different properties when they reach a certain organelle
Endomembrane System
Endoplasmic reticulum
Golgi complex
Endosomes
Lysosomes
Endoplasmic Reticulum
Rough endoplasmic reticulum (RER)
Smooth endoplasmic reticulum (SER)
lumen
or cisterna
7
8
9
- Rough ER (pancake shaped): Ribosomes (rough part) are placed on the ER.
- The ribosomes inject proteins inside the ER
- compartment inside the RER is the lumen or cisterna of the ER
- Smooth ER (tubular shape) : the inside of the ER is never in contact with the cytoplasm.
- environment inside is different from the environment in the cytoplasm
- the mitochondria and chloroplasts are outside of the innermembrane system.
SER Functions
Synthesis of steroid hormone
Detoxication Oxygenases
Cytochrome P450
Glucose release
Sequestering Ca
++
Rough Endoplasmic Reticulum
Protein secretion
Lumen proteins
Membrane proteins
Membrane
Protein
Synthesis
10
11
12
- proportions hange in different tissues
- detoxification in liver : oxidize
- cytochrome P450 (oxygenase) makes hydrophobic molecules hydrophillic
- Ca++ is important for cell signalling
- lumen is inside
Methodology
Pages 267-273
(section 8.2)
Cell
Fractionation
Microsomes
Cell
Fractionation
Microsomes
13
14
15
- should read sec. 18.4 - labratory tecniques
- cell fractionation : two approaches
1. use a microscope to look at a single cell itself
2. take a whole bunch of cells, mash them up, seperate comonents and analyze seperately
- take cells and put them in a tube with buffer (liquid thats isotonic), use a plunger thing fits inside tube with the cells
and buffer. smash cells approx 100. times on ice.
- take homogonate and spin it in a centrifuge at a med. speed. Have a seperation of the different compartments of
the cell. Mito, nuclei go to the bottom of the tube
- transfer supernatant to a new tube, spin at much higher speeds using an ultracentrifuge (50,000 G for 2 hr)
- seperate what is left (postmicrosomal supernatant and microsomes. In the microsomes, you rupture the
membranes of the cell components, most of these membranes are ER)
- possible to see that proteins are made in two seperate places in cells :
- cytoplasm and ER
Secretion Pathway
Cell fractionation Autoradiography
Pulse chase
Autoradiogram
of
pulse chase
experiment
We can gure out where proteins travel by doing
pulse-chase experiments in combination with
cell fractionation or autoradiography
Thats great.
But how do we gure out how proteins actually get secreted?
16
17
18
- can be used with the previous technique
- pulse chase experiment: to figure out how cells get secreted and which places they pass by to go out of the cell
- they gave the cells media that contained radioactive amino acids (cystines), because sulfur in cystines can be
radioactive (have a radioactive isotope). The cystine then gets incorporated into the newly made proteins. the
protines that have a cystine will have the radioactive a.a
- red dots are newly made proteins that have been incoroporated in golgi complex that are labelled with radioactive
isotopes. (pulse when they give radioactive to cells) (chase when they give normal cells) - all the other proteins that
are made after that wont have radioactive. The newly made proteins : 3 min still in ER. 20 min: go to golgi
- found that all protiens go to golgi before they go out of the cell
- once you do pulse chase, you can do 1. cell fractionation or 2. autoradiography (you look at the individual impact
cells.
- printed film using radiographic emultion
- take fixed cells in their plates and cover the plates with liquid emultion that contains the silver grains. When
exposed to light or radiation, they become black. You can see that some parts of the cells will have the silver grains
that turned black .... it shows where the proteins have travelled to. ER > GOLGI > ENDOSOMES > proteins leave
- used yeast cells. With the yeast cells, the were able to make mutations in certain proteins by treating the cells with
certain chemicals (mutagens). They looked at different strains of yeast cells that had mutations, some ofthem had
problems with protein secretion. They screened mutations to see which proteins had problems with protein
secretion. Able to isolate secretion mutants (SEC MUTANTS ). Screened by doing the pulse chase experiment and
autoradiography on thousands of strains to find the mutants that had problems with the secretion pathway. Called
SEC mutants. They were classified in 5 different classes
Yeast SEC Mutants
C
E
D
B
A
So, an experiment would
go like this.....
Grow temperature sensitive mutant yeast and non-mutant control
at normal temperature (room temp)
Pulse chase
Autoradiography
Raise temperature to activate mutation (about 36
o
C)
Observe under microscope
Endomembrane System
Endoplasmic reticulum
Golgi complex
Endosomes
Lysosomes
Plasma membrane
19
20
21
- CLASS A: proteins didn't leave the cytoplasm, didn't even enter ER. Found that The gene that's mutated is
probably a gene that encodes the proteins that's necesarry for the newly made protein (nascent protein) to enter the
ER. accumulate in the cytosol
-CLASS B: Nasecnt proteins aren't getting in ER, they accumulate in RER, but can never leave. Probably whatever
protiens that are important for nascent to go to golgi aren't functioning.
- CLASS C: proteins accumlate from ER > golgi transport vesicles.
- CLASS D: accumulation in golgi
-Class E: accumulation in secretory vesicles... - had to select for mutants that they can still grow, like yeast. If
mutations are always active, the cells would just die. They made mutants without having cells die because the
mutants are heat-sensitive mutations. They are called that because the mutations that they introduced make some
of the proteins more fragile, but still works at normal growth temperature. When you raise the temperature, the
protien starts to unravel and denature. That's why the mutation is heat sensitive.
1. need a control, always grew mutant proteins with a control cell (no mutation) at room temp
2. raise temp to activate. For yeast, 36 degrees is warm
3. pulse chase
4. autoradiography
5. observe
- probably did a thousand times + - able to isolate the gene

2 biosynthetic
(secretory)
pathways

Constitutive secretory

Regulated secretory
(storing in secretory
granules)
Endocytic pathway
vesicle transport

lumen = inside of vesicle = outside of cell

Summary

The endomembrane system comprises the

smooth ER, rough ER, golgi, lysosomes,
endosomes

Several techniques and systems have been

developed to study vesicle/protein transport:
examples: pulse-chase (analysis by membrane
fractionation and autoradiography), yeast mutants

2 biosynthetic pathways: constitutive

secretory, regulated secretory
22
23
24
- 2 biosynthetic pathways found:
1. constitutive
2. regulated
- both are the same all the way till the vesicles that pass the golgi. The vesicles are regulated differently
1. vesicles go automatically to the outside of the cell. Not regulated
2. vesicles are called secretory granules, they accumulate right underneath the surface, when the proper signal is
given, they then expell their content outside (ex. synapses)
3. endocytic pathway: cell will take things inside. The way the cell regulates what's on its surface
- the lumen of any compartment(ER, golgi, lysosomes) is always seperate from the cytoplasm, even when you have
a vesicle forming that takes in materials, the green is never in contact with the white. The receptors always maintain
the same orientation.
- membrane that points towards cytoplasm iis always going to be the same
- membrane that faces towards lumen is always going to be the same membrane
- when a vesicle fuses to the plasma membrane and expels its content, its the luminal side of the vesicle that gets
expelled. Membrane inside the lumen eventually becomes the outside leaflet of the plasma membrane.
-
There are many ways for proteins
to move between cellular compartments
We know that vesicles
shuttle material between
compartments...
but how do proteins get
incorporated in these?
from Lodish et al., MCB
Some ribosomes are attached to the ER and some are not
Ribosome
new
protein
25
26
27
- different ways for proteins to move around in the cell
- proteins getting shuttled are part of the membrane of the vesicle
-ribosomes translate information from mRNA into protein
- makes new proteins that are incorporated inside the lumen of the ER
A very important doodle!!!
from Gnter Blobel Nobel Lecture, December 8, 1999
FIRST PUBLISHED IN 1971
The Signal Hypothesis
1. Ribosomes are all the same
2. Its a signal on the protein that tells it where to go
Blobel in 2008
(Wikipedia)
Ribosomes are all the SAME
(no special type for proteins destined to cytosol, nucleus, ER...)
ER lumen
Free
ribosomes
Membrane
bound
ribosomes
An important experiment
Protein made in vitro
Protein made in vivo
Blobel and Sabatini deconstructed the cell and isolated the parts
necessary for in vitro translation. This was key to proving their
hypothesis.
28
29
30
- subunit pool of ribosomes... they clamp onto the mRNA
- can have alot of ribosomes clamping onto the same mrna
- called polyribosomes
- had to deconstruct the cell, take parts that are necessary for invitro translation
- mRNA, ribosomes, tRNA
- made protein invitro, bigger than ones made in vivo..... even though they're the same protein because protiens
made in vitro had a single sequence. In vivo the single seqcence gets cleaved offf. The difference in size is the
single sequence
The signal peptide is the postal code of the protein
(sequences FYI)
Cotranslational
Translocation
Mechanism
Signal Sequence Hypothesis
GTP Signal
peptidase
BiP Calnexin
HOLD!
See Fig 8.12
31
32
33
- some for import and export.... etc.
- higlighted in yellow are hydrophobic amino acids
- ribosome clamps onto mRNA, tRna comes and add amino acids as mrna is being read
- signal seqcence gets recognized by SR particle (composed of 6 protein)
- SR particle recognizes amino acids and clamps onto the amino acid
-SRP receptor faces towards the cytosol and clamps onto the SRP
- GTP used as a molecular fuse (timer). When SRP becomes active, it binds to GTP. Proteins that bind to GTP are
called GTP ases
- GTP then gets hydrolied to GDP and this allows the SRP particle to leave
- nascent proteins bind to chaperones to allow the protein to fold properly without any interference from the other
protiens. Signal peptidase cuts of signal peptide
Protein synthesis on the rough ER

Includes: secreted, lysosomal, membrane bound, reside in

ER or golgi
1. translation starts on a free ribosome
2. signal peptide holds translation and SRP binds to signal pep.
3. SRP docks the ribosome to a SRP receptor (both bind GTP)
4. SRP release and move to translocon
5. translocon plug is removed by elongating peptide, signal peptide
is cleaved by a signal peptidase
6. Chaperones bind to elongating peptide and ensure proper
folding
Problem:
How do proteins get
integrated into the
membrane?
The signal-peptide does not have to be at the beginning!
fromAlberts et al., MBC
Start-transfer sequence
sequence
34
35
36
4. SRP moves to transolocon and gets released. Once get released, translation can continue
- differenceis that signal peptide is internal and doesn't get cleaved off
- internal start-transfer sequnce is hydrophobic, It's borderd by negative and postive charged amino acids
- positivley charged a.a are in the cytosol side
From http://bcs.whfreeman.com/lodish6e/
go to: animations > select Chap 13 > select Synthesis of...
The signal sequence can snap into the translocon in the other direction
depending on how the protein is encoded
(this orients the protein the correct way)
fromAlberts et al., MBC
Membrane proteins can have many many transmembrane domains...
3
+
-
in
out
...But all proteins use the same basic mechanism to get in
(eg. a voltage gated sodium channel)
37
38
39
-
Double-pass transmembrane protein
fromAlberts et al., MBC
STOP-
transfer
START-
transfer
Mature
protein
NH2 COOH
+ - + - + - + -
+ + + +
- - - -
NH2 COOH
start
transfer
stop
transfer
start
transfer
stop
transfer
cytosol
ER lumen
Membrane
Lipid
Synthesis
40
41
42
- two types of internal signals that don't get cleaved off. They both become the transmembrane domains of the
protein
- can have multiple start transfer and stop transfer
- has four transmembrane domains
- stop transfer is a point where there's a signal where the ribosome is told to stop to thread the protein through
EXOPLASMIC
LEAFLET
EXOPLASMIC FACE
CYTOSOLIC FACE
CYTOSOLIC
LEAFLET
OR LUMEN
Membrane Synthesis and Asymmetry
Membrane
Synthesis and
Asymmetry
Leaet
Cytosolic
Exoplasmic
Clicker Quiz #5
Will be on...
From Membrane Lipid Synthesis
to (but not including) the slide on
Targeting and Vesicle Fusion
43
44
45
- cytosolic leaflet faces the cytosol
-exoplasmic leaflet faces either the lumen ofa vesicle or the outside of a cell
- keep same orientation throughout the biosynthetic pathway (until reaches the surface of the cell)
- membranes maintaing symmetry
Membrane Lipid Synthesis
from Lodish
LUMEN
CYTOSOL
1. Phosphoipids are inserted into
pre-existing membranes (in the ER)
2. Phospholipid synthesis occurs
on the cytosolic face
3. Flippases are needed for
transfer to exoplasmic face
Modication by enzymes
Vesicle formation
Phospholipid
transfer
proteins
Why is the lipid composition of organelles different?
Summary

membranes arise from pre-existing membranes

(components are added in the ER)

Asymmetry is maintained

Synth of phospholipids occurs on the cytosolic face and

are transfered to the exoplasmic face via a ippase

Lipids are then carried from the ER to the Golgi via

transport vesicles

The action of enzymes (in the lumen), differential

inclusion of lipids in vesicles and phospholipid transfer
proteins produce membranes with different compositions
in different organelles
46
47
48
- there's a cytosolic side and a lumen side. The ER is where the lipids get made on the cytosolic side. The enzymes
have their active sites pointing towards cytosol where action happens. Once the phosopholipids are made on
cytosolic side, they need flippase to flip to exoplasmic side.
- lipids are made in pre-existing membranes
- intitially lipid membranes are similira. The structures can be transformed because there are vesicles (lysosomes)
that don't have the same structure of certain organelles.
- need mechanism to change structure after lipids are made : 1) modification by enzymes change lipids after they
are made. 2) Preferential inclusion or exclusion of certain lipids into vesicles and carry them to another organelle. 3)
Phospholipid transfer proteins, where enymes pluck certain lipids from one membrane andput it inside another
membrane.
- before the lipids go to the plasma membrane, they go through the golgi
Post-translational Protein
Modications
Disulde bond formation
Folding
Proteolytic cleavage
Multimer assembly
Glycosylation
N-linked
O-linked
Glycosylation
Glycosyltransferase
Nucleotide sugar
Carbohydrate chain
49
50
51
- disulfide bond formation: bonds formed between cystines
- folding: folding of the protein occurs during translation, but mostly afterwards
- proteolytic cleavag: sometimes smaller proteins are made as larger protein. cutting up enzymes
- multimer: some proteins can have more than one subunits
- glycosylation:
- n or o linked. O-linked is more common.
- n-linked is called n because they are linked on the asparagine. First sugar monomer side chain attached to
asparagine is N-Acetyl.....
- when sugars are being built, oligosaccharides (10-30 monomers) made from precursors that are attached to the
nucleotide carrier. This carrier allows the monomer to get recognized by the enzyme. The enzyme that builds the
sugar trees are called glycosyltransferase (transfers sugar to new growing tree)
N-linked Glycosylation
Dolichol
LUMEN
CYTOSOL
Oligosaccharide-
protein transferase
Protein
Quality Control
Quality
control
Monitoring
Enzyme
UGGT
Calnexin
or
Calreticulin
52
53
54
1. N-linked occurs at the endoplasmic reticulum. the sugar residues don't get added immediately to the protein. We
need a holder (special lipid called dolichol). initially the glycosolations add the first few monomers to the dolichol
initally. The initial carbs (oligosaccharides) point towards the cytosol.
2. special kind of flippase that flips the dolichol towards the inside of the lumen
3. oligosaccharide protein transferase enzyme, The oligosaccharide continues to grow until its ready to be plucked
from dolichol and added to growing peptide.
- consensus sequnce for addition of n-linked is asparagine, and any a.a and either a cyrine or thyrine, than the
enzyme (oligosaccharide-protein...) plucks one of the sugar trees being mae and adds it to protein. Protein can
have several oligosaccharides
N-linked glycosylation summary
- occurs at the ER. Entire oligosaccharide is made before being added to nascent peptide.
- special lipid, dolichol, acts as a holder for the growing carb branches
- glycosyltransferases add monomers to branches
- carb faces cytosol during early synthesis. Then flipped to lumen
- oligosaccharide protein transferase transers oligosaccharide to peptide
- protein quality control: once proteins are made, they don't behave how we want them to
- when protein is made, chaperones are bounnd to the hydrophobic sites.
- unfolded protein, glucosidase (enzymes that cleave glucose) are recognized by an enxyme called calnexin
(membrane bound) or calreticulin (both chaperones) they recognize oligosaccharide by binding to the glucose and
gives protein a chance to fold properly. After some time the protein leaves. If protein is properly folded it will be able
to exit the ER. If not correctly folded, hydrophobic a.a. point towards outside (not properly orientated have to point
inside). UGGT recognizes hydrophobic a.a that aren't oreintated properly in protein. Protein not properly folded is
recognized by UGGT protien or other enzyme (it sends the unproper protein back to translocon, sent to cytosol
(reverse translocation). Protein that isnt properly folded bumps into UGGT. another glucose is added to another
oligosaccahrdie, recognize by calnexin, another chance to fold properly. Then detach and goes through process
again. If not able to fold properly, it'll be sent towards the cytoplasm
Proteasome
Sec. 12.7
Pg. 541-542
E1, E2, Ubiquitin ligase (E3)
ATP
Unfolded
Protein
Response
Nucleus
ER
Membrane
ER lumen
cytosol
55
56
57
- two main players in regards to proteins that are sent back to cytosol through translocation. Proteasome is the
protein destroyer. It chews up proteins, these proteins first have to be tagged. The tags that are placed on proteins
that are meant to be destroyed. They are smal protines (8.5 kDa) added to other proteins to mark them for
destruction. Called ubiquitin
- 3 enzymes that recognize proteins that are supposed to be destroyed. Necessary for adding ubiqutiin. E1 AND E2
recongize the proteins, recognizes hydrophobic poining to the outside. E3 called ubiqutin ligase (something that ties.
The E3 will work with E1 and E2 to add ubiqutin on the protein.)
- can have several that are added back to back
- E1 & E2 bound to hydrophobic side of protein. E3 recognizes and adds ubiquitin
- proteolysis occurs in Beta
- occurs when there is stress, like heat (causes protein to unfold/denature). Cell evovled this mechanism to keep its
proteins folded properly
1. alot of unfolded protein, transmembrane protein sensors sense proteins that are being misfolded (when there is
no stress, the chaperones bind to sensors weakly.When there are a lot of misfolded, they want to bind more to
hydrophobic sites. Chaperones on sensors act as inhibitors) Gets activated by forming pole dymers. Two kinds of
sensors: inactive sensors and activated sensors. One sensor acts as a kinase. 2. translation factor when
phosphorolated binds to the protein 3. makes more mRNA for proteins like chaperones and other proteins capable
of removing stress.
If nothing
works....
Apoptosis
(programmed
cell death)
Summary

In the protein Quality Control process, the calnexin (or

calreticulin) chaperone uses glucose as a marker for folding
while the GT protein detects misfolded proteins and adds a
glucose if another round of folding is needed

If the misfolded state persists, the protein is sent to the cytosol by

reverse translocation (through the translocon) where the
protein is polyubiquitinated and degraded by the
proteasome.

If proteins cant be sent to the cytosol quickly enough, the

Unfolded Protein Response comes into play. Gene expression
of chaperones, coat proteins and QC proteins is increased while
overall translation is decreased.

If this does not work still, the cell commits suicide (apoptosis).
Endoplasmic
reticulum
Golgi
Intermediate
Compartment
(ERGIC)
Endoplasmic
reticulum
Golgi
Intermediate
Compartment
(ERGIC)
Vesicular
tubular
cluster
(VTCs)
58
59
60
- for well folded proteins
- transported to another organelle called the golgi apparatus. After protein translation, post-translation modification occurs inside the golgi.
- Transition stage between ER and golgi
- vesicles are formed and are coded with proteins
- ERGIC area between the ER and golgi (area). There are vesicles in ERGIC called vesicular tubular clusters VTC
- VTC become the golgi and they bud from the rough ER and fuse into the VTC to become the golgi
Camillo Golgi
Invented silver-based
histological stains
Allowed the visualization of
many new cellular structures
1906 Nobel Prize in Medicine
for the study of structures of
the nervous system
Golgi Complex
Cisternae
Golgi Complex
61
62
63
- developed silver based stains because silver binds to proteins.
- cis-Golgi network is where things enter. They exit at the trans-golgi.
- as there are more and more mebranes full of proteins to the VTC, it fills in the holes in the golgi so that you no longer have tubules. The
stack of tubules is being built underneath and move. The ones being made are the new ones
- cis cisternae have a lumen in the middle
Stack of
electron
micrographs
3-D tomographic reconstruction of a Golgi apparatus
The seven cisternae that
comprise the Golgi
CIS
C1, light blue;
C2, pink;
C3, cherry red;
C4, green;
C5, dark blue;
C6, gold;
C7, bright red
TRANS
The ER (yellow) forms
a single continuous
compartment within
the modeled region,
traversing the Golgi
ribbon at multiple
points, extending in
opposite directions
beyond the cis-most
and trans-most
cisternae
64
65
66
-
N-linked Glycosylation in the Golgi
Glycosyltransferases
(EXAMPLE ONLY, STRUCTURE AND ENZYME NAMES FYI)
CIS TRANS
Anterograde
Retrograde
Evidence for cisternal maturation

New proteins being secreted are never seen in vesicles,

they remain in the cisternae.

The only vesicles seen travel in a retrograde direction

(from trans to cis) and contain Golgi-resident enzymes.
67
68
69
1. enzymes placed in different stacks in the cisterna. In cis golgi you have the alpha, trims sugars
reason you have a lot of sacs is for modification to happen
- probelm with anterograde: didn't see nascent proteins travelling in the anterograde direction. You should see the
nascent proteins in the vesicles. Instead the membranes of the trans-golgi go backwards in the retrograde
direction.The newly made proteins remained in the cisternal sacs.
- cisternal maturation model: new proteins that are being secreted are never seen in vesicles while in the golgi
because they remain in the cisterna. The only vesicles seen that travel are travelling in the retrograde direction. This
is because
- cisternal maturation model: new proteins that are being secreted are never seen in vesicles while in the golgi
because they remain in the cisterna. The only vesicles seen that travel are travelling in the retrograde direction. This
is because.
- a
From http://bcs.whfreeman.com/lodish6e/
go to: animations > select Chap 8 > select Protein secretion
Animation:
Protein transport through the Golgi apparatus
Vesicle Transport
Role of protein coats:
1. Mechanical device
2. Selects components
to be carried
COPII-coated forward from ER to
ERGIC and Golgi
COPI-coated retrograde from
ERGIC and Golgi to the ER and from
trans Golgi to cis
Clathrin-coated TGN to endosomes
and lysosomes; plasma membrane to
cytoplasmic compartments
Coated Vesicles
70
71
72
- resident golgi eznymes are what stays in the golgi. They are brought back to the newly made golgi
- vesicles are made on the ER and needed to be coated by special proteins called protein coats. they act as 1. a
mecahincal device: by binding to the membrane, it curves the membrane to make the vesicle that will bud off the
ER. 2. selects components to be carried, selects proteins that are supposed to go to the next compartment.
- three types of protein coats:
1. COP2: used for making movement go forward from ER to ERGIC to Golgi
2. COP1 : for retrograde movement
- everything that happens after the golgi, is mediated by clathrin coat:
Coated
Vesicles
COPI
COPII
Clathrin
COPII-coated Vesicle Assembly
Sec24p
Disassembly: Hydrolysis of GTP on Sar1
73
74
75
-
- need coat proteins to form a protein coat. Also need a nucleating protein that initiates the formation of the coat. For
COP2 it's called Sar1, which uses GTP as a timer like the SRP receptor. Whenever sar1 binds to GTP it's able to
bind to COP1 proteins. The COP proteins stick to the cargo receptors and form the coat on the cytosolic side. Golgi
enzymes are newly made transmembrane proteins that need to be brought to the golgi. Cargo binds to cargo
receptors. The coat proteins are going to bind to the part of the receptor that sticks out on the cytosolic side. The red
nascent proteins need to be secreted and go to the golgi, The coat then has to fall off. Once the vesicle has budded
off, you don't need the coat anymore. Happens when GTP is hydrolized to GDP. Vesicle then gets transported to the
next compartment using motor proteins.Sec24p is a coat protein found in mutants (gives name to protein). There are
several diff kinds of cop2 coats, that select for different content. All called cop2 beause they go from er to golgi. Once
the vesicle has been made and is budded off, the coat proteins fall off, and other proteins replace the coat proteins
COPI Coated Vesicles
Retrograde transport
Trans to cis direction in Golgi
ERGIC and Golgi to RER
ARF1
Retrieval
Signals
KDEL
lys-asp-glu-leu
(soluble prot.)
Binds to KDEL receptor
KKXX
lyslys-X-X
(membrane prot.)
Binds to COPI
If this protein gets accidentally sent to the Golgi, it
will be transported back to the ER
76
77
78
- mechanism to bring back proteins is based on cop1 (trans to cis direction)
- same as cop2 with the exception that the nucleating protein that binds the coat is ARF1 instead of sar1
- formation of the coat is the same, but the proteins that are ER residents have a retrival signal on them which is
alwalys the same (KDEL). The chaperones have the KDEL proteins.
-KDEL proteins all bind to KDEl receptor.
- KDEL sequence at the very end. It binds to a receptor that has the sequence KKXX, those proteins are going to
get recognized by cop1 proteins.
Targeting of ER synthesized
Proteins
Lysosomes
Acid hydrolases
Proton pump
H
+
-ATPase
Autophagy
Autophagolysosome
Residual
body
Targeting of
Lysosomal
Enzymes
Mannose
79
80
81
- lysoomes are ful of acid hydrolases that cut up proteins, sugars etc.
- called acid hydrolases because they are active in an acidic environment,
- what causes the acidity are other proteins called proton pumps,
- major role of lysosomes in cells:
- process of what lysosomes do.
- Autophagy means to digest the old organelles that are no longer useful
- ex. mitochondria that are old get digested inside the lysosome. ER forms around the old cell and form and
autophago lysosome so it can digest the bbig organelles. When it's digested, a residual body is left. Some of the
material gets expelled out of the cell. Sometimes we keep the materials and they're called lipofuscin. Old people
have lot of lipofuscin in retina compared to young
- how do we send the bad enzymes to the right place? Because of the carbohydrates.
- some sugar monomers are called mannoses
Glycosylation of Lysosomal
Enzymes
Cis Golgi
Mannose-6-phosphate
Targeting of
Lysosomal
Enzymes
Mannose
Clathrin
Formation of Clathrin Coated Vesicles
82
83
84
- cisterna of the cis golgi
- a lot of these enzymes are trans membrane.
- N-acetyl... enzyme needs to see that there's a signal on the actual peptide, which says its meant to be sent to the
lysosome. When it recognizes it, it will get some sugars (UDP, nucelotide sugars). Also a phosphate is added
between sugar and monnose residue in position 6, that;s why its mannose-6-phosphate.
-
- in the cis golgi, the protein is tagged with the phosphae will get recognized by a receptor called mannose-6-
phosphate receptor. that occurs at trans golgi
- the protein coats that are made in the trans golgi are based on clathrin
- clahtrin forms a coat
- arf1 protein ,adaptor protein makes lin between the receptor
- once clathrin leaves, coat is shedded, rab replaces
Targeting of
Lysosomal
Enzymes
Mannose
Clathrin
Endosome
Targeting
Vesicle Fusion
Rabs
Tethering
85
86
87
- intermediate vesicle called endosome
- how do
- transport vesicle lost its coat and gets coated with rab (gtp bidning protein)
- about 60 diff rabs known
- between transport vesicel and target membrane there's another rab
- first step is vesicle tethering (3 step process)
Rabs specify vesicle destination
(targeting specicity)...
FYI!
Examples of Rabs (source: Wikipedia)
Docking
SNAREs
SNARE
motif
Fusion
NSF
SNAREs
88
89
90
- second step is called docking
- it involves another protein SNAREs
- three diff snares.
- v-snares: vesicle snares
- t-snares: traget snares 1) trans membrane snares 2) peripheral target snares
- purpose of snares is to bring the vesicle as close as possible to the traget membrane. eventually the vesicle gets
so close to target mebrane that they fuse together
- ctyosolic protein NSF (adp depenedent protein) untwists snares so they can be used again
FYI!

Targeting/tethering: Rabs + tethering proteins

Docking and fusion: SNAREs

SNARE dissociation: NSF

Summary
91
92
93
Endocytic
Pathway
Receptor-
Mediated
Endocytosis
Coated
pit
Coated
vesicle
Coated Pit Structure
Exterior of
cell
Interior of
cell
94
95
96
types of endocytosis:
1. receptor mediated : uptake of receptors and any material bound to them
2. phagocytosis: uptake of particulate matter (cell eating)
3. pinocytosis : uptake of solutes (cell drinking)
- autophagy: the process of organelle turnover, is not a type of endocytosis per se but it uses a similar mechanism
to phagocytosis
- endocytic pathway:late endosome, mannose receptors get recycled,
- clathrin is used as a coat to form the vesicles
- coated pit: young vesicle formed at the membrane
- clathrin can self assemble, will form the lattices
Clathrin
Triskelion
Animation of clathrin triskelion
assembly into a lattice
Clathrin coated vesicle
97
98
99
- the shape of the clathrin molecule is called triskelion
- clathrin molecule consists of 6 sub-units: 3 heavy chains, 3 light chains
- form a soccer ball type structure
- inside the cell, there's constraint. clathrin cell forms on surface of plasma membrane
- ap2 is a multi subunit protein
- has gtp binding protein called arf6
AP2
adaptor
Cell
exterior
Cytosol
Dynamin
GTP
hydrolysis
Conformational
change
Endocytic
Pathway
Housekeeping
Receptors
Transferrin
Low-density lipoprotein
Clathrin
100
101
102
- AP2 adaptor recognizes inter-cellular domain of the receptor.
- there's a signal that says the receptor should be taken in
- for signalling receptors, the signal is triggered by binding alot of material : down-regulation of receptors
- once you have formation of the vesicle: - clathrin can' pinch off molecule on its own. Need dynamin (ring like
protein) which forms a structure around the clathrin coated vesicle to pinch it off. Without the vesicle can't pinch off
of the plasma membrane. Dynamin is a GTP binding protein. Binds: when it's bound to gtp, forms ring structure,
need gtp hydrolysis for actual vesicle to pinch off and gets carried to where it's supposed to go. Eventually the
clathrin coat falls off.
experiment: putting a variant of GTPgammaS (doesn't hydrolized to GDP, stays in GTP forever). Noticed that you
get rings (of gtp) forming because it can't pinch off. These are dynamin rings. Non-hydrolizable GTP analog > GTP
gamma S. To ribosomes on ER when add GTPgammaS: they will always stay down to one another. To vesicles:
the coat doesn't fall off, and the vesicle won't be able to bind to its target.
1. housekeeping: receptors that are always brining in material that the cell needs to function normally. Can be used
again. These receptors get recycled, and the decision to recycle or not takes place in the early endosome. They are
vesicular in structure. Seperation of cargo (blue) and house keeping receptor because of pH.
- examples: transferrin > brings iron in cell
low-density lipoprotein > brings cholesterol
Endocytic
Pathway
Housekeeping
Receptors
Transferrin
Low-density lipoprotein
Signaling Receptors
Hormones
Growth factors
Receptor down-regulation
Clathrin
Clathrin
Early
endosomes
Late
endosomes
Endocytic
Pathway
Posttranslational Protein
Uptake
103
104
105
2. Signalling:hormones and growth signals really have to regulate the amount of signal that goes to the cell.
Signalling receptors kill receptors when there's too much.
- late endosome: if sent here, it gets sent to lysosome ... therefore whatever goes there is gone.
- after translation
- about how proteins get taken into the mitochondria
-
Mitochondria
Targeting sequence
Chaperone proteins
Receptors
Energy two sources
Mitochondrial protein import
Hsp60
Proton motive
force
Brownian
ratchet
Biased
diffusion
106
107
108
- blue arrow: materials get taken in after translation
1. targeting: different order of amino acids. The carrier proteins know that it is a protein going to the mitochondria
2. chaperonses are necessary for bringing in protein once its docking on the receptor
- signal sequence for proteins going into ribosome have no charge (hydrophobic)
-here the signal sequnece is + charged and binds to receptor on what is called to TOM complex (trasport
outermembrane complex). The cytosolic chaperones HSp70 bind to the protein that's being transported and use atp
to mainatain the protein unfolded. Once protein binds to receptor : (left side of diagram) - the protein that gets
completed incorporated into the matrix, the TOM complex sits very closely to th TIM 23 complex: pushing in the
protein requires atp, to get the positibly charged tip of th protein past the intermembrane space because it is very
positively charged (proton motor force). Once its past, th proton motor force actsas another energy force to get the
protein in, will repulse positively charged signal (called pre-squence). Once the +charged signal gets passed, you get
miochondrial chaperones. Once protein sticks head through TIM23, mito chap grab the head and brownian motion
happens (molecules vibrate). Protein goes up and down and as soon as it goes down a matrix mito chap binds to it.
Mitochondria
Targeting sequence
Chaperone proteins
Receptors
Energy two sources
109
- brownian ratchet moves in one direct. Once chaperone binds to it, it can't go back. Because it moves in one
direction the protein diffues, but more in one direction than the other (biased diffusion). Once protein is all in mito
processing peptidase finds sequnce. Right side: internal signal sequence that once it gets into TIM22, it will diffuse
and will become part of the membrane
2. chaperones are not just for folding.
3. receptors on the TOM complex
4. ATP and proton motive force for energy