test 4 Flashcards
1
Q
What is the pulse –chase experiment?
A
Pulsed the proteins with leucine in a pancreas cell and “chased” them to see the pathway they took for secretion
2
Q
What pathway did Jamieson and Palade discover?
A
The Secretory Pathway
3
Q
Why do they observe silver grains in the mitochondria Or in the ER at later time points?
A
Because proteins that don’t remain in the cytoplasm are targeted to the mitochondria and/or ER?
4
Q
What are yeast secretory mutants?
A
Where proteins were transported to is identified by 5 classes of yeast secretory “SEC” mutants that block secretion of newly synthesized protein at 1 of 5 steps (classes A-E)
5
Q
How did the yeast sec mutants help understand the mechanism of protein transport?
A
Helped understand how trafficking vesicles fuse because mutants accumulate secretory vesicles
6
Q
How was the mechanism of protein transport uncovered?
A
By conducting experiments using isolated rough ER microsomes
7
Q
What are microsomes? Polysomes? And Stripped microsomes?
A
Microsomes: Rough ER broken up into vesicles (baby ERs)
* Polysomes: cluster of ribosomes held together by a strand of mRNA
* Stripped microsomes: microsomes with polysome peeled off of it
8
Q
What experiment demonstrated that proteins are inserted into the lumen of RER co-translationally?
A
1) isolate rough microsomes from cells making them a single protein
* 2) peel off the polysomes from the microsomes using puramycin
* 3) perform protein synthesis cocktail (in vitro translation) to identify synthesized products
9
Q
What is the signal sequence?
A
Piece of mRNA that directs the ribosome to the ER
10
Q
What is a nascent peptide?
A
Chain that is still attached to the ribosome before membrane attachment
11
Q
What was the experiment used to demonstrate that signal sequence is cleaved co-translationally?
A
- 1) Isolate stripped off polysomes
- 2) use as substrate for in vitro (protein cocktail) in conditions that only the nascent chains will be able to complete
- 3) analyze the products by SDS PAGE autoradiography (AR) at various times
12
Q
How would you interpret the Gel from Blobel and Dobberstein (1975)
A
- If removal of signal sequence was post-trans, then just the UPPER BAND will be present
- The darker the band, the proteins fell off
- Larger sized proteins will start to appear on the top band
- Post translational: just the upper band appears suggesting newly
synthesized proteins still have the signal sequence attached
13
Q
Why is there a strong lower band?
A
The signal sequence has already been cut off the mRNA strip prior to the start
14
Q
Why do you see an upper band in later time points?
A
Newly synthesized proteins still have the signal sequence attached to them
15
Q
What would be gel look like if the signal sequence was cleaved post-translationally?
A
Just UPPER BAND will be present
16
Q
What are the key players of Ribosome-ER docking mechanism?
A
- Signal sequence emerges form ribosome as protein is synthesized
- SS binds Signal Recognition Particle (SRP)
- SRP binding stops protein synthesis
17
Q
What is the function of SRP?
A
- To bind signal sequence to SRP receptor in the ER membrane
- Halt translation (binding of SRP to signal peptide causes a pause in protein translation)
- GTP binding/hydrolysis
18
Q
What is the function of the signal sequence?
A
- An amino acid chain located on the N-terminus (the start) of proteins and gives a signal on where that protein is supposed
to be translated in the cell (to the ER in this case)
o Sort of like a ticket to a destination
19
Q
How can you experimentally demonstrate that the signal sequence on a secretory protein is necessary for targeting to and
insertion into the RER?
A
Clone, then exclude the amino acid tick (SS), use green fluorescent protein (GFP) to visualize it
20
Q
How can you experimentally demonstrate that the signal sequence on a secretory protein is sufficient for targeting to and
insertion into the RER?
A
Transect cells with a cDNA encoding a chimeric protein that encodes a cytosolic protein but with the signal sequence at its N-terminus
Result: the chimeric protein will be targeted into ER and actin will be secreted
21
Q
What happens if you remove or add signal sequence (SS) from proteins?
A
- Addition of SS: cytoplasmic protein targets protein for secretion from cell (to be taken out of the cell)
- Removal of SS: protein normally secreted will remain in the cytoplasm
22
Q
What is the Mechanism of SS and SRP recognition and binding
A
- SRP finds the SS
- SRP then binds the ribosome holding the SS to the SRP receptor on the ER membrane
- SRP then connects the SS to the transcolon in the ER membrane
23
Q
What is a transcolon?
A
The channel inside the ER membrane that the protein is translated into
24
Q
Is the transcolon open or closed if there is no translocating protein?
A
- The transcolon is sealed with a plug when not translocating protein
- Transcolon has a narrow neck in channel to prevent movement of ions through channel when plug is removed
25
How would you test the transcolon model?
* 1) purify rough microsome (mini rough ER)
* 2) lipids will fill in the hole
* 3) add rough microsomes, put into chamber
* 4) rough membrane and absorb via symbiosis
* 5) measure the conductance (current passing through the membrane)
26
What is the secretory pathway?
* The movement of proteins from the ER -> Golgi -> secretion vesicles
* ER synthesis of lumen and integral membrane proteins (IMP)
27
What are integral membrane proteins (IMP)? How are they inserted into the ER?
* IMPs are integral membrane proteins that are permanently attached to the ER membrane
* Inserted via translocation
28
What are the different types of IMP?
type 1-4
29
How is type 1 inserted into the membrane
One transmembrane domain, N-terminus on the Lumen side
30
how is type 2 inserted into the membrane
Internal SS-spanning transmembrane sequence, N-terminus on the Cytosol side, (+) charges on the cytosol side
31
how is type 3 inserted into the membrane
N-terminus on the Lumen side, no SS
32
how is type 4 inserted into the membrane
no SS, 2 membranes at the same time in the ER meaning both N and C-terminus in the cytosol
33
Can IMP insertion also be post translational? What is the mechanism?
* Yes, protein enters ER after complete synthesis of protein in its unfolded state
* The signal sequence (SS) is cut off by ER signal peptidase
34
What are the different co and post translational modifications /processes taking place in the RER?
* 1) co-translational cleavage of N-terminal signal sequence
* 2) co-translational transfer of N-linked core-oligosaccharide (glycosylation) to nascent N-X-S/T sequences on emerging
protein
* 3) glycosylation, and undergo folding issues
* 4) co and post translational formation/breakage of disulfide bonds by protein disulfide isomerase
* 5) initial post-translational trimming of N-linked core oligosaccharide
* 6) post-translational completion of ternary and quaternary protein structures
35
What are the different steps of O-linked glycosylation? Which steps are co-translational and which ones are post?
Oxygen-linked glycosylation is post-translational and catalyze glycosylation reaction
36
What is the function of glycosylation?
* Proteins must be protected because the sugars around the proteins are being cut (proteolysis)
* Required for proper folding of proteins
* Lysosome is modified to a ticket
o Canberequiredforpropertargetingaftersynthesis
* Can contribute to biological activity of mature protein
37
How are proteins folded?
Chaperones
o BiP
o PDI:proteindisulfideisomerase
38
What are chaperons?
Assist proteins with folding properly
39
What is the function of BiP and PDI, Calnexin and Calreticulin proteins?
* BiP: allows antibody to be folded correctly to then be secreted to the vesicle
* PDI: breaks sulfide bonds to refold and break the bonds of proteins
* Calnexin and Calreticulin:
o o o
bindcarbohydratechains aidinfoldingofglycoproteins
bind to misfolded proteins and destroys them so they can’t be transported to the Golgi complex
40
What diseases maybe cause by improper protein folding?
* Hereditary emphysema: amino acid changes
* Cystic Fibrosis
41
What is the structure, function of Golgi?
* Flattened stacks of cisterna (pita breads)
* Packages synthesized proteins into budding vesicles to be secreted
42
What is the default pathway of protein transport?
Anterograde: ER -> Golgi -> plasma membrane
o Proteins go out of the ER
43
What is retrograde protein transport?
Retrograde: Golgi allows ER proteins to be brough backwards back to the ER (because they are resident ER proteins)
o Retro literally means backwards in Latin
44
How would you use pulse-chase to observe functions of different Golgi compartments?
* 1) pulse chase labeling with different sugars attached to both O-linked and N-linked oligosaccharides revealed differential
labeling of Golgi stacks
* 2) differential localization of Golgi enzymes based on histochemical deposition of reaction products
* 3) Immunolocalization of different resident Golgi proteins suggest discrete compartments
45
What are the functions of different Golgi stacks?
* Cis: beginning (where the ER is connected to)
o Phosphorylationofoligosaccharidesonlysosomalproteins
* Medial: middle
o AdditionofGlcNAc
* Trans: end (where vesicle budding takes place)
46
What are the two models for cargo transport through the Golgi?
* Static: cisterna in the Golgi don’t move, proteins move via vesicles to each “static” cisternae
* Maturing: cisternae are moving form cis -> medial -> trans through the Golgi
47
What are the pros and cons of both? (static and maturing)
* Static: can only move smaller proteins
* Maturation: can only move bigger proteins
48
What might be the actual model? (what is the trend these days?)
Model might be a fusion of both static and maturation methods
o Golgi uses static for moving small proteins and uses maturation for moving larger proteins
49
What are recycling and exocytic routes that proteins travel on?
* ER -> Golgi -> Plasma membrane
* ER -> Golgi -> secretory storage vesicle
* ER -> Golgi -> endosome/lysosome
* ER ->Golgi (for resident Golgi proteins)
* Golgi -> ER (for resident ER proteins: retrograde)
50
What are transport vesicles? What are their functions?
The little circles (packages) that carries the proteins form one destination to another
51
What are coat proteins? What is their function?
* coat proteins: protein shell covers
o Clathrin:coatstheVesicles o COP1:coatstheGolgi
o COP2:coatstheER
52
How can the cell selectively sort cargo into different pathways?
There must be sorting signals in the little tickets (signal sequences) that tell the proteins which pathway to take
53
Why are sorting signals important?
* They “tell” the proteins where to go
* Proteins contain specific ER exit signals that concentrate proteins in vesicles to be exported to ER by COP2 coats
54
How do vesicles bud and fuse with the target membrane?
* 1) lumen facing receptor binds cargo protein
* 2) Cytoplasmic domain of receptor recruits coat protein
* 3) coat effects formation of transit vesicle
* 4) transit vesicle dock/fuses with appropriate acceptor
membrane
55
what are SNARES?
Specific donor and acceptor proteins that effect donor/target membrane recognition and fusion
56
What are some examples of sorting mechanisms?
1) Retrieving resident ER lumenal proteins and membrane proteins
* 2) retention of resident Golgi stack membrane proteins
* 3) selective delivery of lysosomal enzymes
57
What is the function of COPI and COPII proteins? (where do they mediate transport from to where)
* COP1: forms return vesicles that recycle ER proteins with KDEL sequence back to ER, mediate a retrograde transport
* COP2: coats the ER, are required for selective export of newly synthesized proteins from the ER to the Golgi
58
What are important sorting signals?
KDEL
59
What is importance of KDEL sequence?
KDEL sequence are attached to resident ER lumenal proteins that lets the Golgi know to send it back to the ER
60
What is the significance of having different pH in different cell compartments?
Once the ER resident proteins become acidic (move away from basic ER) the KDEL sequence will bind to KDEL receptor in
the Golgi near the C-terminus
o C-terminusistheendofaproteinsequence
61
What would happen if you removed KDEL sequence from a resident ER protein? And if you add KDEL sequence to a secretory protein?
* If KDEL was not present in the ER protein it will get secreted out of the cell because it doesn’t have the “ticket” to go back to the ER
* If KDEL was added, the protein will have the “ticket” needed to be transported back to the ER
62
What is the sorting signal for lysosomal proteins/enzymes?
M-6-P
63
What are the two models for Resident Golgi proteins to sort into correct cisternae?
* Bilayer Thickness (Golgi-Locks Model)
* Kin Recognition
64
What is the endocytic pathway?
Moving membrane and materials inside of the cell
65
What is nonspecific endocytosis and receptor mediated endocytosis?
* Nonspecific endocytosis (Pinocytosis): unspecific intake of extracellular fluids
* Receptor-Mediated Endocytosis: intake of specific extracellular ligands after binding to receptors on membrane
66
What is phagocytosis?
The intake of particulate matter
67
What are clathrin coated pits?
* Substances that enter the cell through receptor-mediated endocytosis (RME) become bound to coated pits on the
membrane
* Clathrin-coated regions turn inside out into the cytoplasm and then pinch free of the cytoplasm
68
What are different steps leading to phagocytosis?
* Step 1) Entrapment: pseudopods surround the particle
* Step 2) Engulfment: cell takes in the particle
* Step 3) Digestion: lysosomal digestive enzymes break down the particle
* Step 4) Absorption: food nutrients in the particle is absorbed
69
What type of materials enter through phagocytosis? What is their fate?
* Large types of particles are taken in the cell using phagocytosis
* The plasma membrane takes up a particle and pinches off to form a phagosome
* The phagosome fuses with a lysosome and the material is digested within the phagolysosome
70
Which bacteria can survive phagocytosis?
* These bacterium species hijack the phagocytic machinery for their own survival:
o Mycobacteriumtuberculosis o Coxiellaburnetti
o Listeriamonocytogenes
71
What are the major organelles of endocytosis?
* Endocytic vesicle
* Early endosomes and late endosomes
* Lysosome
* Trans-Golgi network (recycling endosome)
72
What is the endocytic pathway?
When vesicle bound materials brought into the cell are transported in vesicles and tubules (endosomes)
73
What is the significance of pH in endosome maturation?
* Early endosome (endosomes that have just entered the cell) have a higher pH (more basic)
* Late endosome (endosomes that are further into the cell) have a lower pH (more acidic)
* The further the endosome is into the cell and the more acidic it is, the more maturation the endosome have undergone
74
Why is the endosome recycling important? What happens to the material not recycled?
* Some internalized components are recycled back to the plasma membrane for reuse, this allows the cell to maintain
organelle identity
* Material that doesn’t get recycled go to the lysosome
75
What is a lysosome? What is its function?
To break worn-out cell parts
76
What is the function of transport vesicles / coated pits?
Substances that enter the cell through receptor-mediated endocytosis (RME) become bound to coated pits on the
membrane and transported by transport vesicles
77
What is the structure of clathrin coated vesicle?
* Looks like a soccer ball
* Primarily hexagonal structures with some pentagons
78
What is other machinery involved in clathrin mediated endocytosis?
* 1) receptors with cytoplasmic adaptor targeting domains
* 2) adaptors that bind to receptor target domains
* 3) clathrin triskelion to form clathrin “basket” /coated pits
* 4) dynamin to effect scission of coated vesicle
* 5) uncoating chaperones to remove clathrin coat
79
What are the steps of Clathrin coated vesicle formation?
* Adaptor proteins bind globular head of clathrin head of clathrin heavy chains
* Mediates interaction between clathrin coat and cytoplasmic domain of receptor
80
What is the signal recognized by adaptin complex?
tyrosine
81
What is the function of dynamin?
* Wraps around and forms neck of clathrin coated vesicles
* GTPase activity associated with pinching off vesicle
82
What is the mechanism of vesicle fusion? (what is early and late endosome?)
Primary endosome fuses with sorting vesicle (late endosome)
83
How can you visualize each step of endocytosis?
GFP (green fluorescence protein) used to tag proteins to see them in fluorescence
84
Describe the role of GFP in visualizing proteins involved in endocytosis
* Little green balls turn red by the cell membrane in the picture
* Green = beginning of endocytosis when the clathrin (green) starts coating the vesicle entering the cell
* Red = clathrin (green) leaves, and actin (red) arrives to vesicle to start pushing it inside the cytosol
* GFP is also used to build a timeline of the clathrin coating
85
What are different receptors taken in through clathrin mediated endocytosis?
* Non-recycled receptors: receptors degraded by lysosome to not be used again or “recycled”
o Ligandbindingstartsendocytosis
o Growth factor receptors
* Recycled Receptors: receptors returned to plasma membrane for a new round to be “reused and recycled”
o Nutrient ligand receptors
o LDL(cholesterol)receptor
* Other CME (clathrin-mediated endocytosis) Cargos: tricks the receptor to bring something in, for example viruses use this
to trick host cell into letting it in
86
What is the role of pH?
* When/how ligand binds to a receptor depends on the right pH to do so
o LDL binds at a pH of 6, Iron at 5.5 and so on
87
What are the steps of LDL transport through CME (clathrin-mediated endocytosis)? What are the key proteins involved and what is the function of each?
* 1) LDL particle linked to ferritin
* 2) LDL particle binds to cell surface receptors
* 3) LDL clusters over the coated regions of the membranes
* 4) Brought in the cell via vesicles
* 5) Then transported to the Lysosome
88
What is LDL and why is it important?
Because it needs to be broken down in the lysosome
89
Why does LDL needs to end up in the lysosome?
To be broken down and prevent excess LDL in the bloodstream with causes high blood pressure
90
What happens to the receptor?
The receptors are recycled and brought back to the plasma membrane for reuse
91
What is the important sorting signal present on the LDL receptor?
NPxY
92
How would you interpret the graph of LDL uptake and kinetics?
* At the beginning of the experiment, the mg of LDL is high at the binding of
the Plasma membrane because that is what happens first
* mg of LDL then increases during internalization because they are brought inside the cell (internalized) and put into endosomes
* Endosome then fuses with lysosome and LDL begins to break down (degradation) (see graph)
93
Could you use Pulse chase to visualize the LDL uptake and kinetics?
Yes, radioactively label LDL and Pulse Chase it through the Endocytic Pathway
94
How would different mutations affect LDL uptake and kinetics?
* 1) Null Mutation: no LDL surface binding, therefore no internalization can occur without the LDL able to bind to the surface
* 2) Mutated Ligand binding domain: No LDL or internalization, therefore the receptor localizes to coated pits
* 3) Truncated Cytoplasmic Domain: LDL binds to the cell’s surface BUT no internalization, therefore the LDL gets stuck at
the surface with no way in (no sequence)
* 4) Stop Codon Mutation before Transmembrane Domain: No binding and No internalization, therefor the LDL can’t attach
anywhere to begin with
* 5) Y à X Mutation in Cytoplasmic Domain: LDL binds to the surface BUT no recruitment to coated cells
o This mutation messes up the clathrin that recruits the adaptor, so the LDL gets stuck at the plasma membrane
95
Why would mutations at step of LDL uptake or mutation in LDL receptor lead to heart disease?
* Mutations in LDL or it’s receptor result in high levels of LDL in the bloodstream which then leads to heart disease
o Hard for the heart to pump blood if excess LDL is accumulated in the bloodstream
96
What is the statin-fiber treatment? How to they promote LDL uptake?
* Stain fiber treatment is meant to increase the cell’s ability to intake (uptake) LDL to be broken down by the lysosome
o This is not used for LDL mutations, just for regular people with high blood pressure
* Promotes LDL uptake by:
o 1)stopping the cell’s ability to produce its own cholesterol(LDL) but shutting off the pathway
o 2)tricks cells into thinking they need to bring in more cholesterol to breakdown LDL
o 3)dietary fibers that binds to bile acids and prevents recycling
