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test 4 Flashcards

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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

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2
Q

What pathway did Jamieson and Palade discover?

A

The Secretory Pathway

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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?

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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)

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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

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6
Q

How was the mechanism of protein transport uncovered?

A

By conducting experiments using isolated rough ER microsomes

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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

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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

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9
Q

What is the signal sequence?

A

Piece of mRNA that directs the ribosome to the ER

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10
Q

What is a nascent peptide?

A

Chain that is still attached to the ribosome before membrane attachment

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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
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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
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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

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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

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15
Q

What would be gel look like if the signal sequence was cleaved post-translationally?

A

Just UPPER BAND will be present

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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
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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
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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
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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

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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

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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
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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
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23
Q

What is a transcolon?

A

The channel inside the ER membrane that the protein is translated into

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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
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25
Q

How would you test the transcolon model?

A
  • 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)
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26
Q

What is the secretory pathway?

A
  • The movement of proteins from the ER -> Golgi -> secretion vesicles
  • ER synthesis of lumen and integral membrane proteins (IMP)
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27
Q

What are integral membrane proteins (IMP)? How are they inserted into the ER?

A
  • IMPs are integral membrane proteins that are permanently attached to the ER membrane
  • Inserted via translocation
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28
Q

What are the different types of IMP?

A

type 1-4

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29
Q

How is type 1 inserted into the membrane

A

One transmembrane domain, N-terminus on the Lumen side

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30
Q

how is type 2 inserted into the membrane

A

Internal SS-spanning transmembrane sequence, N-terminus on the Cytosol side, (+) charges on the cytosol side

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31
Q

how is type 3 inserted into the membrane

A

N-terminus on the Lumen side, no SS

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32
Q

how is type 4 inserted into the membrane

A

no SS, 2 membranes at the same time in the ER meaning both N and C-terminus in the cytosol

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33
Q

Can IMP insertion also be post translational? What is the mechanism?

A
  • Yes, protein enters ER after complete synthesis of protein in its unfolded state
  • The signal sequence (SS) is cut off by ER signal peptidase
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34
Q

What are the different co and post translational modifications /processes taking place in the RER?

A
  • 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
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35
Q

What are the different steps of O-linked glycosylation? Which steps are co-translational and which ones are post?

A

Oxygen-linked glycosylation is post-translational and catalyze glycosylation reaction

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36
Q

What is the function of glycosylation?

A
  • 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
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37
Q

How are proteins folded?

A

Chaperones
o BiP
o PDI:proteindisulfideisomerase

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38
Q

What are chaperons?

A

Assist proteins with folding properly

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39
Q

What is the function of BiP and PDI, Calnexin and Calreticulin proteins?

A
  • 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
Q

What diseases maybe cause by improper protein folding?

A
  • Hereditary emphysema: amino acid changes
  • Cystic Fibrosis
41
Q

What is the structure, function of Golgi?

A
  • Flattened stacks of cisterna (pita breads)
  • Packages synthesized proteins into budding vesicles to be secreted
42
Q

What is the default pathway of protein transport?

A

Anterograde: ER -> Golgi -> plasma membrane
o Proteins go out of the ER

43
Q

What is retrograde protein transport?

A

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
Q

How would you use pulse-chase to observe functions of different Golgi compartments?

A
  • 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
Q

What are the functions of different Golgi stacks?

A
  • Cis: beginning (where the ER is connected to)
    o Phosphorylationofoligosaccharidesonlysosomalproteins
  • Medial: middle
    o AdditionofGlcNAc
  • Trans: end (where vesicle budding takes place)
46
Q

What are the two models for cargo transport through the Golgi?

A
  • 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
Q

What are the pros and cons of both? (static and maturing)

A
  • Static: can only move smaller proteins
  • Maturation: can only move bigger proteins
48
Q

What might be the actual model? (what is the trend these days?)

A

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
Q

What are recycling and exocytic routes that proteins travel on?

A
  • 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
Q

What are transport vesicles? What are their functions?

A

The little circles (packages) that carries the proteins form one destination to another

51
Q

What are coat proteins? What is their function?

A
  • coat proteins: protein shell covers
    o Clathrin:coatstheVesicles o COP1:coatstheGolgi
    o COP2:coatstheER
52
Q

How can the cell selectively sort cargo into different pathways?

A

There must be sorting signals in the little tickets (signal sequences) that tell the proteins which pathway to take

53
Q

Why are sorting signals important?

A
  • 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
Q

How do vesicles bud and fuse with the target membrane?

A
  • 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
Q

what are SNARES?

A

Specific donor and acceptor proteins that effect donor/target membrane recognition and fusion

56
Q

What are some examples of sorting mechanisms?

A

1) Retrieving resident ER lumenal proteins and membrane proteins
* 2) retention of resident Golgi stack membrane proteins
* 3) selective delivery of lysosomal enzymes

57
Q

What is the function of COPI and COPII proteins? (where do they mediate transport from to where)

A
  • 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
Q

What are important sorting signals?

A

KDEL

59
Q

What is importance of KDEL sequence?

A

KDEL sequence are attached to resident ER lumenal proteins that lets the Golgi know to send it back to the ER

60
Q

What is the significance of having different pH in different cell compartments?

A

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
Q

What would happen if you removed KDEL sequence from a resident ER protein? And if you add KDEL sequence to a secretory protein?

A
  • 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
Q

What is the sorting signal for lysosomal proteins/enzymes?

A

M-6-P

63
Q

What are the two models for Resident Golgi proteins to sort into correct cisternae?

A
  • Bilayer Thickness (Golgi-Locks Model)
  • Kin Recognition
64
Q

What is the endocytic pathway?

A

Moving membrane and materials inside of the cell

65
Q

What is nonspecific endocytosis and receptor mediated endocytosis?

A
  • Nonspecific endocytosis (Pinocytosis): unspecific intake of extracellular fluids
  • Receptor-Mediated Endocytosis: intake of specific extracellular ligands after binding to receptors on membrane
66
Q

What is phagocytosis?

A

The intake of particulate matter

67
Q

What are clathrin coated pits?

A
  • 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
Q

What are different steps leading to phagocytosis?

A
  • 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
Q

What type of materials enter through phagocytosis? What is their fate?

A
  • 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
Q

Which bacteria can survive phagocytosis?

A
  • These bacterium species hijack the phagocytic machinery for their own survival:
    o Mycobacteriumtuberculosis o Coxiellaburnetti
    o Listeriamonocytogenes
71
Q

What are the major organelles of endocytosis?

A
  • Endocytic vesicle
  • Early endosomes and late endosomes
  • Lysosome
  • Trans-Golgi network (recycling endosome)
72
Q

What is the endocytic pathway?

A

When vesicle bound materials brought into the cell are transported in vesicles and tubules (endosomes)

73
Q

What is the significance of pH in endosome maturation?

A
  • 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
Q

Why is the endosome recycling important? What happens to the material not recycled?

A
  • 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
Q

What is a lysosome? What is its function?

A

To break worn-out cell parts

76
Q

What is the function of transport vesicles / coated pits?

A

Substances that enter the cell through receptor-mediated endocytosis (RME) become bound to coated pits on the
membrane and transported by transport vesicles

77
Q

What is the structure of clathrin coated vesicle?

A
  • Looks like a soccer ball
  • Primarily hexagonal structures with some pentagons
78
Q

What is other machinery involved in clathrin mediated endocytosis?

A
  • 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
Q

What are the steps of Clathrin coated vesicle formation?

A
  • Adaptor proteins bind globular head of clathrin head of clathrin heavy chains
  • Mediates interaction between clathrin coat and cytoplasmic domain of receptor
80
Q

What is the signal recognized by adaptin complex?

A

tyrosine

81
Q

What is the function of dynamin?

A
  • Wraps around and forms neck of clathrin coated vesicles
  • GTPase activity associated with pinching off vesicle
82
Q

What is the mechanism of vesicle fusion? (what is early and late endosome?)

A

Primary endosome fuses with sorting vesicle (late endosome)

83
Q

How can you visualize each step of endocytosis?

A

GFP (green fluorescence protein) used to tag proteins to see them in fluorescence

84
Q

Describe the role of GFP in visualizing proteins involved in endocytosis

A
  • 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
Q

What are different receptors taken in through clathrin mediated endocytosis?

A
  • 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
Q

What is the role of pH?

A
  • 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
Q

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?

A
  • 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
Q

What is LDL and why is it important?

A

Because it needs to be broken down in the lysosome

89
Q

Why does LDL needs to end up in the lysosome?

A

To be broken down and prevent excess LDL in the bloodstream with causes high blood pressure

90
Q

What happens to the receptor?

A

The receptors are recycled and brought back to the plasma membrane for reuse

91
Q

What is the important sorting signal present on the LDL receptor?

A

NPxY

92
Q

How would you interpret the graph of LDL uptake and kinetics?

A
  • 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
Q

Could you use Pulse chase to visualize the LDL uptake and kinetics?

A

Yes, radioactively label LDL and Pulse Chase it through the Endocytic Pathway

94
Q

How would different mutations affect LDL uptake and kinetics?

A
  • 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
Q

Why would mutations at step of LDL uptake or mutation in LDL receptor lead to heart disease?

A
  • 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
Q

What is the statin-fiber treatment? How to they promote LDL uptake?

A
  • 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

Cell bio (1 decks)

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