Post-midterm content Flashcards
1
Q
Autoradiography?
A
- Take your live tissue/sample and treat it with tritium tagged amino acids
- Then treat the tissue like an normaly EM sample - embed it with plastic and slice into thin sections
3.Next, place photosensitive emulsions over the sample and place it in adark room, allows radioactivity to expose emulsions - The sample is then treated with a developer which shows wherever the radioactive tags were by tiny silver grains. The grains can be seen on an EM to show exactly where the radioactive molecules ended up in the cells.
2
Q
Pulse of the pulse chase experiment?
A
Expose the pancreatic cells to radioactive tritium-labeled leucine for three minutes. This will label all proteins made during this time. When observed on EM they are all in the Golgi
3
Q
Chase #1 of the Pulse-Chase?
A
Chase the sample by not adding tritium-labeled leucine for 7 minutes. After, the 7 minutes the proteins are now found in the Golgi on the EM
4
Q
Chase #2 of the pulse-chase?
A
Chase the sample for 37 minutes total, now the proteins are in vacuoles
5
Q
Chase #3 of the pulse-chase?
A
117 minutes now the proteins are at the plasma membrane waiting for secretions stored in zymogen granules since they are pancreatic enzymes
6
Q
Steps in vesicular trafficking?
A
- Sorting of cargo
- Budding and separation from the source membrane
- Transfer to destination
4.Storage - Recognition of target membrane and fusion
7
Q
COPII location?
A
Found on the ER membrane used for transport from ER to Golgi
8
Q
COPI location?
A
Found on the VTCs and Golgi used for trafficking from the Golgi to the ER and potentially for intragolgi trafficking
9
Q
Clathrin coat protein location?
A
Found on the trans Golgi, found on the plasma membrane and found on endosomes
10
Q
Caveolin coat protein?
A
Found on the cell surface
11
Q
Retromer coat location?
A
Found on endosomes
12
Q
Motor proteins?
A
Used for vesicles that need to go a long distance
Kinesins: move toward the puls ends of microtubule (to cell peripheries)
Dyneins: move toward the minus ends of microtubules (to the golgi)
Myosins: move along actin and only used for short distances
13
Q
Tethering proteins?
A
Tethers: allow vesicles to dock with the target membrane
SNAREs : allow vesicles to fuse with target membrane
14
Q
Characteristics of the golgi?
A
Polarity
Cis-Golgi
Medial-Golgi
Trans-Golgi
Made up of cisternae
15
Q
Anterograde trafficking?
A
From the ER to Golgi
16
Q
Retrograde trafficking?
A
From the golgi to the ER
17
Q
Differential centrifugation?
A
- First you break open cells to release their internal parts into a homogenates
- Then centrifugate the homogenate at different speeds. This separates cell parts bade on their size and shape
- Large particles with large sedimentation coefficient settle into the pellet faster than small particles
18
Q
Sedimentation coefficient?
A
Each particle/organelle has a sedimentation coefficient, which tells us how fast it settles when spun
19
Q
Density gradient centrifugation?
A
- Prepare a test tube filled with a density gradient( Put 45% sucrose solution in the bottom of the tube, then 35% sucrose on top of that , then 25% sucrose and then 10% sucrose(forms a step gradient)
Light liquid is always at the top of the tube and dense liquid is always at the bottom of the tube) - Then we add our sample and spin, particles will move through the gradient to the point where their density is equal to that of the gradient
20
Q
COP II coat formation?
A
- Sar1-GDP in the cytoplasm is activated in Sar1-GTP by Sec12(GEF).
- Sar1-GTP then inserts into the membrane of the ER and recruits Sec23/24
- Sec23/24 can bind to transmembrane cargo with FF signal at C-terminus or DXE or cargo receptors
- Sec13/31 are then recruited and cause the vesicle to curve and cause a conformational change in Sec23/24 leading to hydrolyzation of Sar1-GTP
21
Q
After COP II buds off the ER?
A
- Sar1-GTP hydrolyzes and the coat comes off the vesicle
- Tethering proteins called p115(require Rab1) on two COPII vesicles bind to each other and bring the vesicles close
- The vesicles then fuse forming the VTC right outside the ER
22
Q
How does the VTC get to the Golgi?
A
By binding to dynactin which is bound to dynein this transports the VTC toward the Golgi where they fuse with the golgi using p115
23
Q
Cargo signals recognized by the coat proteins?
A
- COPII: binds to C-terminal FF or DXE found on golgi bound proteins
- COPI: binds to KK for ER proteins
24
Q
pH in ER vs Golgi/VTCs
A
ER: pH is neutral
Golgi/VTCs: pH is slightly acidic
25
ERGIC53
Cargo receptor that binds cargo in the ER lumen with N-linked glycosylations and has a KK signal to come back to the ER and FF signal to leave the ER
-Binds cargo at neutral pH and high Ca2+ and releases it a acidic pH/low Ca2+
26
KDEL receptor?
-Binds to cargo in the golgi/VTC lumen and brings them back to the ER
-Binds cargo in acidic pH and releases them a neutral pH
-Binds KDEL sequence of ER resident proteins
27
P24?
Has both FF and KK signals and binds GPI-anchored proteins
28
Phenotypes of observed yeast with mutant secretory genes?
Yeast had proliferated ER
Yeast with enlarged golgi
Yeast with accumulated vesicles
29
Two types of retrograde transport?
1. COPI dependent
2.COPI-independent: involves formation of tubular transport intermediates
30
COPI transport from Golgi to ER?
1. Arf1-GDP is recruited by a GEF to the Golgi/VTC membrane
2. Beta/gamma are then recruited which bind transmembrane cargo with KK signal
3. Beta/Gamma then recruit alpha/epsilon and beta prime which force the curvature of the membrane and vesicle formation and budding
3. Arf1 GAP then hydrolyzes Arf1 causing uncoating
31
What is moved back to the ER?
-ER cargo receptors
-SNAREs
-Enzymes
-Misfolded proteins
32
Three different models for intra-golgi trafficking?
1. Cisternal maturation: cis becomes the medial cisterna and then the trans cisternae
2. Cisternae are separate compartments and then vesicles were trafficked between the cisternae
3 Golgi were interconnected and then molecules could just diffuse through the golgi
33
Rothman experiment goal?
believed cargo was transported between cisternae by coated vesicles
Reconstitute intra-golgi transport in vitro and identify the molecular machinery, like coats, responsible for the cargo trafficking.
34
What is needed for a cell free intragolgi traficking system?
Purified Golgi membranes that must have retained function(this was done by membrane fractionation)
Cytosol
ATP and GTP
Assay system: to measure transport and determine efficiency
35
Rothmans Golgis ?
1. He used a donor golgi which was from virus-infected CHO cells that expressed the G-protein, but lacked GlcNAc transferase and an acceptor protein that was from normal CHO cells with GlcNAc transferase, but no G-protein.
36
G-protein?
_integral membrane protein
-Travels via the secretory pathway
-Has COP II export signal
37
Logic of Rothman assay?
Mix both Golgi types, add cytosol, GTP, ATP and radioactive GlcNAc
If the G-protein receives GlcNAc then transport must have occurred
This means vesicles carried G-protein to the enzyme, or vice versa
38
Electron microscopy in Rothman?
Showed vesicle formation
Without GTP – No vesicles or coats
With non-hydrolyzable GTP – Vesicles formed but stayed coated
39
Identification of the COPI coat?
EM showsed vesicles forming only in the presence of GTP
He then purified these coated vesicles using density gradient centrifugation which lead to the discovery of the COPI coat
40
How were Algal scales evidence for cisternal maturation?
Botanists found that algae produce very large carbohydrate rich scales that are too big to fit inside COPI vesicles, yet these scales still move through the golgi suggesting cisternae must mature to carry cargo forward.
41
G-protein temperature sensitive mutations indicating cisternal maturation?
1. Researchers could control when the protein could leave the ER(only left at permissive temps)
2. They used immunogold labeling and tracked the movement of the G-protein through the Golgi and saw there were only gold particles in one or two adjacent cisternae
If vesicles were transporting the protein, you’d expect it to appear in many cisternae at once
42
Procollagen experiment in favour of cisternal maturation?
Procollagen is a large protein too big for COP I and only exist when ascorbic acid is present
Cells were given ascorbic acid briefly, so procollagen could enter the Golgi, then the movement was tracked.
The procollagen moved from the cis-golgi to the medial golgi and then trans-golgi step by step, one cisterna at a time.
43
Possible ways enzymes are moved throughout cisternae?
1. relocated backwards throughout the Golgi as the cisternae mature
2. Enzymes move within golgi via direct connections, enzyme has the natural affinity for where the substrate is which explains how the enzymes are always in the correct order
44
6 functions of the Golgi?
1. Sequential modification of N-linked oligosaccharides
2. O-linked glycosylations
3. Proteoglycan glycosylation
4. Proteolytic modifications of proteins
5. Sorting of proteins to various destinations(late golgi)
6. Production of secretory granules
45
What is a glycoslyation?
Reaction in which a carbohydrate is attached to a hydroxyl or other functional group of another molecule
46
N-linked glycosylations? -
-Occurs to all proteins of the ER
-Helps protein fold properly
-Potentially a signal to exit the ER
-Added to asparagines
47
How is the N-linked gluycosylation modified in the Golgi?
1. Cis-golgi: some mannoses trimmed
2. Medial golgi: GlcNAc residues are added
3. Trans-Golgi: more sugars are added
48
Endo H enzyme?
Can cleave the N-linked glycosylation, but only in their early, high-mannose form. Once GlcNAc is added in the medial golgi, the glycan is resistant to the enzyme
49
Endo H sensitive protein vs resistant?
If a protein is Endo-H sensitive: it hasn’t made it past the cis-golgi
If a protein is Endo-H resistant: it has reached the medial golgi
50
Where does O-linked glycosylation occur?
In the medial golgi
51
What is an O-linked glycosylation?
Addition of first sugar occurs on a serine/threonine and then a chain of up to 100 sugars are added. These can be modified by sulphating. NOT every protein that enters the golgi is O-linked glycosylated
52
Proteoglycans?
These proteins are heavily glycosylated in the medial golgi and mainly found in the ECM
These proteins are mainly sugars with tiny protein
Ex. Heparan Sulfate
53
T/F: The TGN is more acidic than the medial and cis golgi?
True
54
Activation of Furin in the TGN ?
The low pH of the TGN is essential for activating furin which is a protease that is inactive in the ER but active due to acidic pH in the TGN. Furin helps cleave and activate other proteins in the TGN
55
Lysosomal Enymes in the TGN?
Cleaved in the TGN but stay inactiive until they reach the much lower pH in lysosomes
56
Pancreatic enzymes in the TGN?
Cleaved in the TGN but remain inactive until they reach the alkaline pH of the intestine
57
Protein aggregation of the TGN?
The slightly acidic pH of the TGN also promotes aggregation of some secretory proteins
Ex. Digestive enzymes begin to form dense protein clusters in the TGN. These aggregates get packaged into condensing vacuoles, which later mature into zymogen granules.
58
Types of endocytosis?
Clathrin-mediated
Caveolae
Lipid Raft mediated
Phagocytosis
Macropinocytosis
59
Major endocytic compartments?
Endocytic vesicles (bud from the cell surface, there are various kinds)
Early endosomes
Recycling endosomes
Late endosomes
Lysosomes
60
Early Endosome?
-Midly acidic
-Sorting station
-Tubular and globular domain
-Ligands can detach from their receptor and receptor is sorted into tubular domain which becomes recycling endosome
-Membrane contains Rab5 and EEA1 tether
61
Recycling endosome?
-Once tubule detaches from the early endosome they can be referred to as recycling endosomes and go back to the cell surface
-There is a fast and slow pathway
-Return membrane and most receptors to the cell surface
-Rab 11
62
Late endosome(MVB)?
-Vesicles within itself due to ESCRT
-More acidic and start to have degradative enzymes
-Contain Rab7 which recruits HOPS, dyneins and kinesins
-Membrane proteins targeted for degradation are within it
-Some proteins can cycle between the MVB’s and Golgi(sortillin and M6PR)
63
Lysosomes?
Very acidic, for degradative enzymes to function
High protein concentration (appear dark on EM)
Ability to digest almost anything delivered to them
Many specialized channels in the membrane are used to transport amino acids, monosaccharides, etc. into the cytoplasm to be reused
-membrane contains LAMP and LIMP to protect it
64
If a lysosome were to break open and the enzymes enter the cytoplasm what would happen?
Nothing since the cytoplasm is neutral
65
Vacuolar ATPase?
-Found in all endosomes + Golgi + VTC
-Not in mitochondria
-Pumps H+ ions across the membrane to accumulate inside lysosome
-Can be set to acidify these compartments to a different level
66
Bafilomycin?
inhibits the V-ATPase pump
67
Ionophores?
forms a channel that lets protons leak out of the membrane
68
Weak bases?
grab a proton and cross the membrane with it(ex. Ammonium + chloride), or sop up the proton (ex. chloroquine)
69
Clathrin-mediated endocytosis?
1. Clathrin binds to the membrane and forces it to curve and form a coated pit
2. Clathrin binds to adaptors
Adaptors: equivalent to the inner layer of the COPII coat
These bind the cargo and cargo receptors
3. After the clathrin coated pit has formed dynamin will form rings around the base of the vesicle and pinch off the vesicle
4. Coating and uncoating varies depending on the clathrin vesicle
70
AP-2?
Clathrin adaptor on the cell surface
71
Dynamin?
Polymerizes into rings around the base of the clathrin-coated vesicle, GTP hydrolysis causes the rings to contract which pinches off the clathrin coated vesicle from the PM
If these do not work clathrin coated vesicles are stuck at the PM\
72
Non-clathrin endocytosis?
Macropinocytosis - actin
Phagocytosis - actin
GEEC pathway - lipid raft endocytosis and GPI-anchored proteins
Caveolin-mediated endocytosis
73
What are lipid rafts?
-Microdomains on the plasma membrane
-Made up of two specific types of lipids(cholesterol and sphingolipids) that assemble together
-The lipid raft is thicker and more rigid than regular PM
74
What are cavolae?
-Small-flask shaped invaginations of the PM
-Rich in cholesterol and sphingolipids thus rich in lipid rafts
-Coated with caveolins, integral membrane proteins that help shape and stabilize the pit
-Do not uncoat
75
Transcytosis?
Movement of a substance across a cell from one membrane domain to another
76
Caveolae trancytosis of albumin?
1. Albumin binds to gp60 receptor at the apical endothelia and the caveolae bud off, travel through the cell, and fuse with the opposite membrane to release albumin into tissues
This process is dynamin-dependent
77
Knocking out of albumin?
Albumin accumulates on the cell surface, but does not cross the endothelium. Mice survive by making their capillaries leaky.
78
Caveosome?
When caveolae are endocytosed, they can fuse together to form caveosomes(neutral compartments)
79
Caveolae and cell signaling?
-Serve as signaling pathways
Many signaling molecules prefer raft-like regions, so they gather in caveolae
Some signaling proteins bind to a conserved region on caveolin, increasing signaling efficiency
80
What is macropinocytosis?
Occurs in every cell
Nonspecific endocytosis
Cell extends long lamellipodia outside the cell(actin-dependent) and encloses around ECF and molecules. They lead to the formation of a large vesicle called a macropinosome that will be transported to the lysosome.
81
GPI-Anchored Proteins?
-Concentrate at lipid rafts
-Embeds only in the outer leaflet of the PM and does not span the membrane, anchors the protein at the cell surface
82
Folate receptor?
-GPI-anchored protein
-Folate receptor binds folate at the cell surface and then it is brought into the GEEC pathway
83
What does CLIC-GEEC do?
Internalizes fluid, membrane, and GPI-anchored proteins(often in lipid rafts)
Does NOT use clathrin
84
How does CLIC-GEEC work?
1.CLICs are small tubulovesicular structures that pinch off from the PM and internalize cargo like the folate receptor
2. CLICs fuse together to form larger vesicles called GEECs which are slightly acidic(more than early endosome).
3. GEECs then fuse with early endosome and folate is transported into the cytoplasm via a membrane transporter
85
What is LDL?
LDL carries cholesterol and lipids in the blood
-Contain apolipoprotein B, which sticks out and allows LDL receptors to recognize it
86
Endocytis pathway of LDL?
LDL binds LDL receptor at the plasma membrane(neutral pH)
LDL receptor is recruited into a clathrin-coated pit using the ARH adaptor protein
The pit buds in and forms a clathrin-coated vesicle
Vesicle fuses with the early endosome, which is slightly acidic
Acidic pH causes the LDL to dissociate from the receptor
Receptor recycles back to the surface via recycling tubules
LDL stays in the endosome and eventually goes to the lysosome
In the lysosome LDL is broken down into:
Cholesterol
Fatty acid
Amino acids
87
Endocytosis of EGF and EGFR?
1. EGF binds to EGFR on the plasma membrane, this activates the receptor and triggers endocytosis of the EGF-EGFR complex.
2, The complex is internalized into the cell
3. Unlike the LDL receptors, EGFR does not recycle
4. EGF-EGFR complex stays bound and moves through the endocytic pathway when it reaches the lysosome both EGF and EGFR are degraded
5. This shuts off the signal, preventing the cell from dividing repeatedly
EGFR is degraded as a safety mechanism so that the cell becomes temporarily insensitive to more EGF and prevents uncontrolled growth
88
Iron endocytosis pathway?
1. Transferrin(loaded with iron) binds to the transferrin receptor on the cell surface
2. This complex is brought into the cell via clathrin-mediated endocytosis(uses AP-2 adaptor protein that recognizes tyrosine motif on the receptor)
3. The vesicle fuses with the early endosome(pH slightly acidic)
4. The acidic pH causes the iron to dissociate from the transferrin
5. Iron is transported out of the endosome into the cytoplasm via transporters
6. Transferrin(now without iron) remains bound to the receptor
7. The receptor and empty transferrin are recycled back to the cell surface
8. At the neutral pH of the PM, transferrin dissociates from the receptor and is released
89
Transcytosis of IgA?
1. IgA binds to a receptor on the basolateral surface of the epithelial cells
2. The receptor-IgA complex is internalized
3. The complex travels through endosomes inside the cell
4. It is then exocytosed at the apical surface of the epithelial cell
5. IgA is released into the mucous layer where it binds pathogens and viruses neutralizes/aggregates them for macrophages to eat
90
Two main routes for TGN exit?
1. Secretion to plasma membrane
2. Cargo transported to endosome
91
Constitutive secretion?
: secretion that occurs all the time (continuous, house-keeping secretion, does not respond to signals)(immediate fusion with PM)
92
Regulates secretion?
found in specialized cells(ex. Pancreatic enzymes, neurotransmitters)(here cargo is held near the plasma membrane and only delivered to the plasma membrane when there is a signal)
93
Dense core vesicles?
Found in neurons and endocrine cells contain large neuropeptides(not synaptic vesicles)
94
Secretion in a polarized cell to the apical membrane?
Proteins destined for the apical surface of the epithelial cells are often sorted into lipid rafts before being secreted sorting occurs in the TGN
At the TGN, lipid rafts can cluster into large tubular transport intermediates and help move proteins to the apical membrane
Some of the cargo proteins going to the apical surface will bind kinesins this will put a force on the golgi membrane and pull out a tubule
On the TGN there are dynamin like proteins that will help to cut the tubule off the TGN
Tubular transport intermediates go from TGN to apical surface
95
Secretion in polarized cell to the basolateral membrane?
1. A protein going to the basolateral surface will have a tyrosine-based or dileucine based motif
2. Sorting of the basolateral proteins requires clathrin and uses AP-1B adaptor protein complex
96
Adaptor proteins of clathrin structure?
Each composed of four polypeptides
Bind clathrin through ear domain
All found on the golgi
97
AP-1b, AP-1a, AP-3, AP-4?
AP-1b: form coated vesicles that go to the basolateral surface
AP-1a: involved in retro intermediates that go to endosome
AP-3: involved in targeting from TGN to the endocytic pathway
AP-4: on golgi
98
GGA?
Another type of adaptor protein
Single polypeptide
Has VHS domain to bind KK
Has GAT domain to bind Arf
Binds clathrin via hinge and terminal ear domain
99
Direct vs Indirect transport from the TGN to endosomes?
Direct: TGN to Endosome to Lysosome
Indirect: TGN to Cell surface to Endosome to Lysosome
100
How are membrane proteins of lysosomes transported from TGN to lysosomes?
1. Start in the ER then are transported to the golgi via COPII
2. At the TGN, they are sorted into vesicles destined for endosomes/lysosomes
101
What adaptor protein does LAMP-1 require to get to lysosome?
AP-3, occurs even if GGA is not functioning
102
How are soluble hydrolases transported from the TGN to lysosomes?
1. These are glycosylated in the ER
2. Bind to cargo receptors to leave the ER
3. At the TGN, they bind a different cargo receptor, often M6PR
103
How hydrolases get to lysosomes?
1. Lysosomal enzymes have a specific shape(not sequence) recognized by an enzyme called GlcNAc phosphotransferase in the cis-golgi
2. This enzyme adds a phosphate group to a mannose sugar on the N-linked oligosaccharide which forms mannose-6-phosphate.
3. The M6P prevents further sugar modifications
4. In the trans-golgi, the M6P receptor recognizes and binds the phosphate
5. Uses AP-1a and GGA adaptor proteins to enter clathrin-coated vesicles, these are then carried to early/late endosomes
6. At acidic pH, the enzyme detaches from the receptor and stays in the endosome eventually becoming part of the lysosome
7. The M6P receptor is sent back to the Golgi using a coat protein
104
I-cell disease?
-mutation of GlcNAc phosphotransferase
-M6PR cannot recognize the hydrolase
-Enzymes are mistakenly sent to the plasma membrane and secreted into the blood
-Lysosomes swell with undigested material(inclusions), since they’re missing their enzymes
105
M6P tag formation?
1. In the cis-golgi: GlcNAc phosphotransferase recognizes a signal patch on the lysosomal hydrolase and transfers a sugar-phosphate from UDP-GlcNAc to a mannose on the N-linked oligosaccharide
2. In the medial golgi: Phosphodiester alpha-N-acetylglucosaminidase removes the GlcNAc sugar but leaves the phosphate resulting in M6P
106
Adaptor GGA role?
VHS domain of the GGA recognizes the cargo receptors and binds KK motifs on M6PR or sortilin
GAT domain then binds Arf1-GTP which then recruits the AP-1 adatpors
107
Sortilin?
1. Prosaposin enters the ER, moves through the Golgi, and reaches the TGN
2. Sortilin in the TGN binds to prosaposin which helps package prosaposin into vesicles for lysosome delivery(uses GGA adaptor proteins to form the vesicle)
3. In the lysosome, specific proteases cleave prosaposin into saposin proteins which activates the lipid degrading enzymes
108
Sortilin structure?
Has KK motfis recognized by VHS domain of GGA adaptor
109
Retromer?
Recycles cargo receptor like M6P receptor and sortilin from late endosomes back to hte trans-golgi
Retromer binds specific motifs on the two receptors
110
Retromer structure?
Inner layer: recognizes sorting signals in the cytoplasmic tail of cargo
Outer layer: drives membrane curvature to form the tubule
111
Who does phagocytosis?
Specialized immune cells: macrophages, neutrophils, dendritic cells
112
What is taken up by phagocytosis?
Large particles (bacteria, dead cells, RBCs)
113
Zipper mechanism of phagocytosis?
Sequential engagement of the Fc receptors on macrophages with antibodies pulls the membrane around the bacteria
Forms a phagosome, which later fuses with lysosomes for degradatio
114
Capping in phagocytosis?
If the antibodies are only present on half of the bacteria you don’t get total engulfment(phagocytosis fails)
Antibodies must coat the entire bacteria
115
What happens to the phagosome?
1. The phagosome initially has proteins found in early endosomes (Ex. Rab5), they become early endosome like
2. Then they become late endosome like and then fuse with lysosomes which load it up with degradative enzymes to destroy the bacteria
116
Phagosomal Maturation?
-Phagosomes undergo fusions and protein exchanges first with early endosome then with later endosome
-Once phagosomes have acquired sufficient LAMPs, they begin fusing with lysosomes, this forms a phagolysosome
117
When do we do autophagy?
-Protein aggregates: too large for the proteasome
-Damaged mitochondria: maintain mitochondrial quality(turnover of defective organelles)
-Starvation: recycle non-essential components
-Lipid droplets: release stored energy
-Removal of bacteria/infectious agents
118
Chaperon-mediated autophagy?
Proteins with a KFERQ tag in the cytoplasm bind to Hsc-70 and are translocated to lysosomes via LAMP2A
119
Microautphagy?
Proteins bind to Hsc-70 to phosphatidylserine on the surface of late endosomes are budded into internal vesicles
Internalisation of small regions of cytoplasm
120
Macroautophagy?
large structures inside the cells (proteins/organelles) are enclosed by a double-unit-membrane structure to form an autophagosome
121
T/F: Cargo targeted for autophagy is often ubiquitinated?
True
122
LC3/ATG8 receptor?
Found on the phagophore/isolation membrane, if they bind to something they will start to surround it(oftentimes need to recruit other bilayers0
123
How does macroautophagy work?
1. Autophagy signal triggers formation of the phagophore often ER-derived
2. LC3-I is cleaved and lipidated and becomes attached to PE and forms LC3-II
3. LC3-II inserts into both sides of the growing phagophore
4. LC3-II recruits receptors like P62, which bind ubiquitinated proteins/organelles and brings them into the forming autophagosome
5. Phagophore seals and forms autophagosome which seals with lysosome
Mature similarly to phagosomes by first become early-endosome like then late-endosome like
124
Rab proteins?
-Small GTPases(on = ATP, off = ADP)
-Insert into membrane in GTP state and bind effector proteins
Have fatty acid C-terminus allowing insertion into the membrane
125
The Rab cycle?
1. Rabs are brought to the membranes by GEFs, these are different for almost each Rab(specific for each Rab)
2. Rabs in the GTP form (On the membrane) can bind effectors (different Rabs bind different effectors). Rabs can play a strong role in defining what a membrane is by binding various receptors.
3. Rab GAPs(there are many) inactivate Rabs by causing GTP hydrolysis. This causes the loss of binding to effectors
4. Rab GDI removes Rab-GDP from membranes and sequesters it in the cytoplasm
126
Rab1?
-Found on COPII vesicles
- Rab1-GTP binds a tethering protein called p115(effector)
-This allows the two membranes of the COPII vesicles to tether together and then SNAREs will be recruited to fuse the two membranes together to form VTCs
127
Rab5?
-Found on early endosomes/phagosomes/early autophagosomes
-Tethering protein EEA1
EEA1 requires Rab5 and PI3P to be on the early endosomal membrane
128
EEA1?
tethering protein of Rab5 helps bring early endosomes close together
-Binds to Rab5 and Pi3P
-FYVE domain binds PI3P
-Interacts with SNAREs: syntaxin 6 and 13 for fusion
129
Rab7?
Found on late endosomes/phagosomes/late autophagosomes and lysosomes
130
RILP ?
Effector of Rab7
-binds molecular motors connects late-endosome to dynactin-dynein machinery
131
ORP1L?
Effector of Rab7
- It is a cholesterol sensor, if the cholesterol level is high this will recruit spectrin which recruits dynactin/dynein. Dynein motor will move it toward the minus end of microtubule
132
FYOC1?
-Competes with RIPL for binding, FYOC bind kinesin which leads to movement toward plus end of microtubules
-In low cholesterol FYOC bind Rab7 and late endosome moves toward the plus end
133
HOPS?
Rab7 effector this is the tethering complex that allows fusion of late endosome with each other and with lysosomes
134
Phosphoinositides
Early endosome: PI3P
Late endosome: PI(3,5)P2
135
Maturation of early to late endosome?
1.Change in subcellular localization of endosome(motors)
2. Decrease in pH(late endosomes are more acidic than early endosomes)
3. Change in lipid composition (PI3P becomes PI(3,5)P2)) must have a different kinase
4. Closing off access to the recycling branch
5. Receive acidic hydrolases for lysosome function from TGN
6. Change in fusion machinery (tethers and SNAREs)
136
Conversion of Rab5 to Rab7?
1.Activated Rab5-GTP recruits Rab7 GEF
2. GEF catalyzed the exchange from GDP to GTP on Rab7
3. Activated Rab7 now binds to the membrane of the early endosome and recruits TBC2, which is a Rab5 GAP which catalyzes the hydrolysis of GTP on Rab5 leading to its inactivation it is also removing the Rab5 GEF.
4. GDP bound Rab5 then leaves the endosome membrane and then Rab7 is left creating a late endosome
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Delivery of late endosome to lysosome?
1. Rab7 is found on BOTH late endosomes and lysosomes, so late endosomes and lysosomes can be tethered together by the HOPS complex and Rab7 can also recruit SNAREs to fuse the membranes together
2. Fusion of a late endosome with a lysosome produces a hybrid organelle with characteristics of both, sometimes referred to as an endolysosome
3. The material within the endolysosome is then digested and the endolysosome then becomes lysosome-like again
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Storage forms of lipids?
1. Cholesterol esters
2. Triglycerides
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Cholesterol Esters?
Synthesized by ACAT in the ER membranes (cholesterol + Fatty acid)
Found in the lipid bilayer of the ER
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Triglycerides?
-Synthesized by DGAT1/DGAT2 in the ER membranes
-Typically stores fatty acids
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Two ways of getting triglycerides/cholesterol esters out of ER mem.?
1. Lipid Droplets
2. Lipoprotein particles
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Structure of lipid droplets?
-Cytoplasmic organelles with a monolayer of phospholipids
-Surface proteins(PAT and DGAT2)
-Core;triglycerides + cholesterol esters
-associate with peroxisomes or ER
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All cells can produce LD but what specialized cells produce more?
Adipocytes
Hepatocytes
Mammary glands
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Formation of lipid droplets?
1. Starts in the ER membrane
2. Once LD forms it can still be associated with the ER membrane to
3. DGAT2 can bridge the ER mem. to the LD to allow for direct transfer of triglycerides and cholesterol esters
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ACAT?
Becomes active when there is too much cholesterol in the ER so you make cholesterol esters
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DGAT1?
-Spans the ER membrane multiple time, cannot move into the lipid droplet
-Synthesizes triglycerides
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DGAT2?
-Only goes through ER once so it can move into LD
-New triglycerides made by DGAT2 will be preferentially incorporated into existing lipid droplets reducing ER stress
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PAT proteins(ADRP and Perilipin)?
-Can be phosphorylated by protein kinase A
-Hormone sensitive lipase can bind to phosphorylated PAT proteins and release FA
-Stabalize the surface of LD
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Retrieval of fatty acids from lipid droplets?
1. Beta-adrenergic receptor on the cell surface is activated and releases epinephrine/adrenaline, a coupled G protein cascade is triggered converting ATP to cAMP this leads to activation of protein kinase A
2. Protein kinase A then phosphorylates HSL in the cytoplasm and also some PAT proteins on the surface of lipid droplets
3. Phosphorylated HSL binds phosphorylated PAT proteins which hydrolyzes the triglycerides in the lipid droplet and releases pre fatty acids
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Retrieal of cholesterol from LD?
HSL has some cholesterol esterase activity
Autophagy is the major mechanism for getting cholesterol/fatty acids out of lipid droplets
Lipophagy: if inhibited cholesterol remains stuck in lipid droplets
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How to visualize lipid droplets?
- Fluorescent dyes(Nile red and bodipy) bind to the hydrophobic part of LD
-EM
-GFP-tags associated with LD
-Fluorescently tagged substrates for DGAT1/DGAT2 can also be used
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Structure of lipoprotein particles?
-Forms in ER lumen
-Core: triglycerides/cholesterol esters
-Surface: phospholipid monolayer + Apolipoprotein B
-only made in enterocytes and hepatocytes
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Function?
Allow for transport of lipids between cells in you body
Lipid droplets stay in the same cell they are made in
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Types of lipoprotein molecules?
-Chylomicrons
-VLDL, LDL, IDL, HDL
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Chylomicron assembly in enterocytes?
ApoB48 is transported into the ER. Lipidation then starts in the ER, adding dietary triglycerides and cholesterol esters. More lipids are added in the Golgi. This forms the chylomicron(large lipoprotein particle carrying dietary lipids)
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Secretion and circulation of the chylomicron?
Chylomicrons are secreted into the lymphatics and enter the bloodstream via thoracic duct.
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Triglyceride breakdown of chylomicrons?
Lipoprotein lipase(LPL) on endothelial cells hydrolyze TGs and release free fatty acids for uptake by tissues. This causes the chylomicrons to shrink and become chylomicron remnants
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Chylomicron remnant uptake by liver?
ApoE on remnants allows binding to hepatic receptors; the remnants are then endocytosed by hepatocytes. Remaining lipids and cholesterol are used/stored/ metabolized by the liver
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VLDL?
-Made in liver by hepatocytes
-Repackages cholesterol and triglycerides that the liver endocytosed from chylomicron remnants other apolipoprotein particles or HDL or cholesterol synthesized by the liver
-Contains ApoB100 which can bind LDL receptors but not until TGs are stripped in circulation by endothelial cells that contain lipoprotein lipase
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IDL?
-As VLDL moves through the circulation and loses TGs it becomes IDL
-IDL is VLDL just with the LDL receptor unmasked
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LDL?
-Produced by further stripping of TGs from the IDL particle
-LDL can be endocytosed by most cells of the body
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HDL ?
-Another type of lipoprotein
-ApoA is the major apoprotein in HDL. Addition of cholesterol to ApoA converts it to the HDL particle
-ABCA1 is a transporter that binds ApoA and loads it with cholesterol
-HDL particles are then endocytosed by the liver
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ApoA?
-Found in HDL
-Secreted by the liver
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ApoB48?
-Large
-Found in chylomicrons and lacks an LDL receptor binding domain
-Recognized by receptors on hepatocytes
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ApoB100?
-Large
-Found in VLDL, IDL and LDL particles
-Binds LDL receptors when TGs have been stripped
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Role of the liver: lipid uptake?
-Endocytosis chylomicron remnants
-Collects cholesterol and triglycerides from HDL through reverse cholesterol transport
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Role of the liver: Cholesterol handling?
Can synthesize cholesterol
Cannot break it down
Disposal:
Can convert cholesterol to bile salts(only way to get rid of cholesterol)
Can be used to make steroid hormones
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Role of the liver: Lipid repackaging and storage?
-Cholesterol + triglycerides stored in lipid droplets in hepatocytes
-Can repackage into VLDL
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Role of the liver: Energy release?
-Triglycerides can be broken down into free fatty acids
-Fatty acids can be transported to the mitochondria for ATP synthesis
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Niemann-Pick Transporter?
1. Cholesterol arrives in the lysosome
- Comes mainly from LDL particles , which are broken down in the lysosome
2. NPC2 binds cholesterol in the lysosomal lumen
-NPC2: soluble lysosomal protein. Acts as a shuttle, binding cholesterol in the aqueous lumen and handing it off to NPC1.
3. NPC1 transports cholesterol across the lysosomal membrane (tranmsmembrane protein)
4. StarD4 and similar proteins bind cholesterol in the cytosol
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Niemann-Pick Type C disease?
-Lysosomal storage disease
-Comes from mutations in either NPC1 or NPC2 results in the inability to transport cholesterol out of lysosomes
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Genetic Diseases involving excess LDL?
1. No LDL receptor synthesized
2. Receptor stuck in the ER (receptor synthesize but misfolds)
3. Receptor reaches surface but can’t bind LDL(cannot interact with ApoB-100)
4. Receptor binds LDL but fails to internalize
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Receptor binds LDL but fails to internalize?
Mutation in the cytoplasmic tail of the receptor disrupts interaction with clathrin adaptor, ARH
Involves truncation/mutation of the tyrosine motif
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What happens when you have too much circulating LDL due to excess FA/cholesterol?
1. Cells have enough cholesterol and fatty acids, so they stop producing LDL receptors.
2. This causes LDL particles to circulate and after awhile they start to oxidize(damaged LDL particles)
3. Damaged LDL particles are recognized and eaten up by macrophages, now macrophages are foam cells because they are full of lipid droplets.
4. If this occurs in the SM cells of your arteries this can lead to accumulation of foam cells which results in plaques in the artery walls that can lead to atherosclerosis(heart attack/stroke).
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Gram-negative bacteria?
Ex. Salmonella
Have two membranes and need a more complex system to cross both the inner and outer membranes
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Secretion System III
-Found in Gram-negative bacteria
-A needle structure that spance the bacterial envelope and pierces the host cell membrane to inject effector proteins
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Salmonella infection process?
1. Salmonella attach to the intestinal epithelium by means of adhesins
2. Salmonella is then engulfed by enterocytes and becomes enclosed in an early endosome
3. Normally, early endosomes mature into late endosomes, which then fuse with lysosomes to degrade their contents
4. However salmonella inject SopD2 into the host cell via secretion system III and it binds Rab7
5. SopD2 inactivated Rab7, immobilizing the late endosome and preventing the recruitment of HOPS and fusion with the lysosome
6. Salmonella avoids degradation and the SVC becomes a replication niche for the bacteria
7. Salmonella can then transcytose across the enterocye and reach the submucosa
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Legionella pneumophila?
-Causes legionaires disease
-Can grow in water
-Can cause pneumonia
-Uses type IV secretion system
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Type IV secretion system?
-Found in both Gram positive and negative bacteria
-Can deliver effectors across both the bacterial envelope and the host's membranes
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How does legionella work?
1. Legionella is engulfed by a macrophage into an early phagosome
2. Once inside the phagosome T4SS activates and injects the effector proteins into the macrophage's cytoplasm
3. One major effector ubiquitinates and removes Rab5 from the phagosome membrane. Without Rab5, the phagosome cannot recruit Rab7 and thus cannot fuse to the lysosome.
4.Phagosome remains in an immature "early" state and fails to be able to kill the bacteria
5. The macrophage is now a bacterial incubator where the bacteria can replicate.
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What is a virus?
Protein/lipid with nucleic acids(RNA/DNA and double or single strnaded)
-Dependent on host cell for replication and translation(lack ribosomes)
-Some viruses can replicate on their own (single strnaded)
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Types of viruses?
1. DNA virus
2. RNA virus
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Bacterial infections vs viral?
BActerial: can be treated with antibiotics because bacteria have their own proteins, ribosomes and peptidoglycan cell walls
Viral: Harder to treat since viruses use host cell machinery
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Vaccines?
can be made for both bacteria and viruses but must be tailored specifically to each pathogen
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Non-Enveloped Virus?
Genome: DNA/RNA double or single
-no lipid membrane
-made of a protein shell(capsid) + nucleic acid
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How do non-enveloped cells enter the cells, replicate and exit?
Enter cells by endocytosis
Replication occurs in the cytoplasm
Exit strategy: leave cell by killing the host cell
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Enveloped virus?
Genome:RNA/DNA(double or single)
Structure: surrounded by a lipid bilayer derived from the host cell's membrane
Sensitive to detergents
Viral core with proteins complexed with the viral genome
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Envelope proteins?
Contain integral membrane proteins that cross the envelope and recognize and bind host cell receptors this can permit direct fusion of the virus with the cell’s membrane or fusion after endocytosis
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Life cycle of a virus?
1. Virus attaches to and enters the host cell
Viral genetic material is released into the cytoplasm
2. Early viral proteins are produced
These help with replication of the viral genome, modification of the host cell and formation of “virus factories”
3. Late proteins are synthesized
These are structural proteins that help assemble new virus particles
4. New viral genomes and structural proteins are assembled into virus particles
5. Non-enveloped viruses: released when cell dies and lyses
Enveloped viruses: typically bud off from a cellular membrane, gaining their lipid envelope in the process
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How non-enveloped viruses enter into cells?
Most of the non-enveloped viruses must be endocytosed
They can then either inject their nucleic acid directly into the cytoplasm(polio virus)
Or they can break open that endosome and get their DNA into the nucleus(adenovirus
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Polio virus entry into the cell?
Endocytosis: Poliovirus is endocytosed into the host cell through receptor-mediated endocytosis. The poliovirus attaches to a receptor on the host cell membrane.
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Polio virus RNA translation and replication?
Once the poliovirus RNA genome enters the cytoplasm, it binds to ribosomes, which begin translating the RNA into viral proteins. The viral RNA acts as both mRNA and a template for replication.
The viral RNA contains a ribosome binding site that allows the host ribosomes to immediately start translating the RNA. The virus’s RNA dependent RNA polymerase begins to then replicate the viral genome.
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Replication organelles polio virus?
The virus takes steps to hide its RNA from the host’s immune detection.DsRNA would normally trigger a defense response from the cell, so the virus forms replication organelles that compartmentalize the viral RNA and prevent it detection by the host cell’s immune system.
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How does polio virus modify host machinery?
Ribosome modification: poliovirus modifies the host ribosomes to preferentially translate viral RNA over host mRNA. More viral proteins are made.
Hijacking the Host’s membranes: The virus utilizes host cellular membranes to create structures for replication and assembly, often involving the ER and membrane vesicles
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Polio virus viral assembly?
The newly synthesized viral proteins and RNA genomes are then assembled into new poliovirus particles in the cytoplasm
Poliovirus makes phosphatidylserine rich membranes which will surround clusters of virus particles and these then form autophagosome looking structures. This helps bypass the host cell’s defense mechanisms.
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Exit of the poliovirus?
-Autophagosomal structures fuse with the host cell membrane and release the newly formed viral particles into the extracellular space allowing them to infect nearby cells.
-After the virus, is released the host cell typically undergoes lysis which releases even more viral particles.
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What type of virus is Poliovirus?
Non-enveloped
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Attachment and entry of Sars-CoV-2?
-Spike(S) protein binding: The Sars-Cov-2 virus particle has a surface protein called spike(S) that specifically binds to the ACE2 receptor present on host cells, such as those in the respiratory epithelium and endothelial cells in the blood vessels.
-Conformational change in spike protein: Upon binding to ACE2, the S-protein undergoes a conformational change. This change allows the viral envelope to fuse with the host cell membrane; this enables the virus to inject its genetic material into the host cell.
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Virus entry Sars-CoV-2 via direct fusion?
Viral envelope directly fuses with the plasma membrane of the host cell. This releases the viral RNA directly into the host’s cytoplasm.
200
Virus entry mechanism via endocytosis and fusion with endosome?
Virus is endocytosed forming an early endosome. Once inside, the acidic environment of the endosome triggers a conformational change in the spike protein, leading to fusion of the viral envelope with the endosomal membrane and releases RNA into the cytoplasm.
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Release of viral RNA and translation Sars-CoV-2?
-Once the virus is in the cytoplasm, the nucleocapsid(viral genome wrapped in proteins) is released into the cytoplasm.
-Viral RNA is then recognized by the host cell’s ribosomes because it contains a recognition site. This then translates the viral RNA into viral proteins. Including viral enzymes like RNA-dependent RNA polymerase which is critical for replicating the viral genome.
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Sars CoV-2 modification of the host cell?
Viral proteins synthesis: As the viral RNA is translated into proteins, host cell prioritizes production of the viral proteins rather than its own
Immune Evasion: Sars-CoV-2 has a mechanism to interfere with the host’s immune system, including modifying antigen presentation.
203
Exit of Sars-CoV2?
-Viral particles and genome are then assembled in the cytoplasm often at replication organelles
-Buds out of the cell and takes some the lipid bilayer along with it
204
HIV
The env protein complex of HIV binds to the CD4 protein on the surface of T-cells, and the virus fuses directly with the plasma membrane
205
Influenza?
The HA protein of influenza virus binds to sialic acids on N-glycosylations of cell surface proteins. The virus is then endocytosed and a pH-induced conformational change in HA leads to fusion with the endosomal membrane
206
What are virus factories?
-Specialized structures within infected cells where viral replication and assembly take place
-SARS-CoV-2 produces proteins that insert into the ER membrane and modify it to form specialized structures called virus factories/replication organelles
These contain the dsRNA
This is where replication can occur away from the immune surveillance
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Budding of enveloped viruses?
1. Nucelocapsid assmebly in the cytoplasm
2. VIral envelope proteins are synthesized by host ribosomes and inserted into the ER membrane and transported through the secretory pathway
3. Viral envelope proteins are delivered to the cell membrane, where they become embedded, with their cytoplasmic tails inside the cell
4.Cytoplasmic domains then interact with nucleocapsids in the cytoplasm
5.This interaction triggers budding, where the plasma membrane wraps around the nucleocapsid
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Where does Sars-CoV-2 bud?
Does not bud at the plasma membrane, but rather buds into intracellular membrane (such as VTCs)
One of the envelope proteins(E) acts as an ion channel, collapsing the pH gradient and deacidifying lysosomes
