Endocytosis Flashcards
(209 cards)
1
Q
What are the major endocytic compartments?
A
- Endocytic vesicles
- Early endosomes
- Recycling endosomes
- Late endosomes / multi-vesicular bodies
- Lysosomes
2
Q
What are the different endocytic vesicles?
A
- Clathrin (fuse with early endosomes)
- Non-clathrin (many kinds, mostly deliver their cargos to the same early endosome)
- Phagosomes
- Caveolae-mediated endocytosis.
3
Q
What is the structure of the clahtrin triskelion?
A
A clathrin triskelion consist of 3 heavy chains and 3 light chains.
4
Q
How does clahtrin induce membrane curvature?
A
Clathrin forms a lattice coat that bends the membrane, forming a clathrin-coated pit.
5
Q
How are clathrin triskelions arranged in the lattice?
A
The legs of clathrin triskelions are intertwined with neighboring triskelions to stabilize the lattice structure.
6
Q
What protein in the secretory pathway is functionally similar to clathrin?
A
Clathrin functions similarly to the outer coat of COPII (involved in ER-to-Golgi transport).
7
Q
Does clathrin interact with cargo proteins directly?
A
No — clathrin does not interact with cargo proteins directly.
8
Q
How does clathrin polymerize to form a coat?
A
Clathrin triskelions self-polymerize through interactions between their heavy and light chains.
9
Q
What are the four stages of vesicle formation in CME?
A
(1) Initiation: formation of a clathrin-coated pit, clathrin coat assembly, cargo receptor recruitment.
(2) Propagation: invagination of clathrin-coated pit.
(3) Budding: continual invagination of a clathrin-coated pit, scission of its neck.
(4) Uncoating: disassembly of a clathrin coat.
10
Q
What part of the endocytic machinery does clathrin form?
A
Clathrin forms the outer coat of the vesicle.
11
Q
How does clathrin link to cargo receptors?
A
Clathrin binds to adaptors (mostly AP-2 on the cell surface), which recruit cargo receptors.
12
Q
What is the role of dynamin in clathrin-mediated endocytosis?
A
- Dynamin performs vesicle fission (cuts the vesicle from the membrane).
- Dynamin forms a helix at the stalk of a budding vesicle and uses GTP hydrolysis to constrict the neck and release the vesicle.
13
Q
What is another name for clathrin-mediated endocytosis?
A
Receptor-mediated endocytosis.
14
Q
How do receptors help concentrate cargo?
A
Receptors bind to specific cargo, clustering them in the forming vesicle, which increases efficiency.
15
Q
What are the two main types of clathrin adaptors?
A
(1) AP complexes (Adaptor Protein complexes).
(2) GGA (Golgi-localizing, Gamma-adaptin ear domain homology, ARF-binding proteins).
-> Arf1 (involved in recruitment)
16
Q
What part of clathrin do AP complexes and GGA bind to?
A
Bind to the N-terminal region of the clathrin heavy chain.
17
Q
Which adaptor is important for clathrin function at the plasma membrane?
A
AP-2 (no Arf interaction).
18
Q
What adaptor is used for LDL receptor internalization?
A
ARH (Autosomal Recessive Hypercholesterolemia), which binds to the N-terminal region of the clathrin heavy chain.
19
Q
What biological process is disrupted in shibire mutant flies? What happens with the temperature-sensitive shibire allele?
A
- Synaptic vesicle recycling at the neuromuscular junction.
- At restrictive temperature (30°C) → Flies are paralyzed because synaptic vesicles are not recycled.
At permissive temperature (~20°C) → Paralysis is reversed and flies can move.
20
Q
What is the early endosome’s main function?
A
Acts as a cargo sorting station in the endocytic pathway.
21
Q
Why is the early endosome acidic?
A
The low pH helps to detach ligands from their receptors.
22
Q
What happens to ligands and receptors in the early endosome?
A
- Ligands → Stay in the lumen → Sent to the lysosome for degradation.
- Receptors -> sorted into tubular extensions -> sent to recycling endosomes.
23
Q
What GTPase and tether protein are found in the membrane of early endosomes?
A
- Rab5 (GTPase)
- EEA1 (Tether protein)
24
Q
What are the two domains of the early endosome?
A
- Tubular -> For sorting and recycling.
- Vacuolar -> For cargo degradation.
25
What happens to the tubular extensions of early endosomes?
They detach and become recycling endosomes.
26
What is the difference between fast and slow recycling?
1. Fast recycling → Direct return to the plasma membrane.
2. Slow recycling → Through a recycling compartment near the centrosome.
27
What GTPase is present in recycling endosomes?
Rab11
28
What is the pH difference between early and recycling endosomes?
1. Early endosome -> midly acidic.
2. Recycling endosome -> less acidic.
29
What type of receptors are found in recycling endosomes?
Recycling receptors
30
What is the main function of late endosomes/multivesicular bodies (MVBs)?
Part of the degradative branch - involved in cargo degredation.
31
How is the pH of late endosomes different from early endosomes?
Late endosomes are more acidic than early endosomes.
32
What happens to membrane proteins targeted for degradation in MVBs?
They are budded into intraluminal vesicles by ESCRT complexes.
33
Which ESCRT complexes are involved in inward budding in late endosomes?
ESCRT-0
ESCRT-I
ESCRT-II
ESCRT-III
34
What is not found in late endosomes that is present in early endosomes?
Recycling receptors
35
What proteins cycle between MVBs and the Golgi?
1. Mannose-6-phosphate receptor
2. Sortilin
36
What GTPase is present in the membrane of late endosomes?
Rab7
37
What proteins are recruited by Rab7 in late endosomes?
1. HOPS tethering complex.
2. Dyneins.
3. Kinesins.
38
Do late endosomes contain degradative enzymes?
Yes, but not as many as lysosomes.
39
What is the fate of cargo & membrane proteins in MVBs?
Delivered to lysosomes for degradation.
Membrane proteins targeted for degradation are budded into the internal vesicles.
40
Which proteins can cycle between MVB's and Golgi?
- Mannose-6-phosphate
- Sortillin
41
What is the function of lysosomes in the cell?
Terminal compartment for cargo degradation.
42
What is the pH of lysosomes and why is it important?
pH = 4.5–5.0 → Required for degradative enzyme activity.
43
What kind of population do lysosomes have based on electron microscopy?
Heterologous population of electron-dense organelles, reflecting different stages of cargo breakdown.
44
Why are lysosomal enzymes harmless if the lysosome breaks?
Enzymes (like cathepsin D and acid phosphatase) work only at acidic pH — cytoplasmic pH is too high for them to function.
45
How do lysosomes protect themselves from their own degradative enzymes?
Lysosomal membrane contains highly glycosylated proteins (LAMPs and LIMPs) that protect the membrane from enzymatic damage.
46
What happens to the digested components in lysosomes?
They are shuttled out by specialized channels and transporters.
47
What small GTPase is found on lysosomes?
Rab7 (involved in vesicle trafficking and fusion)
48
Where is the vacuolar ATPase (V-ATPase) found in the cell?
Found in lysosomes, endosomes (all types), Golgi apparatus, and VTCs.
49
Is V-ATPase found in mitochondria?
No, uses a different mechanism for maintaining pH gradients.
50
What is the function of the vacuolar ATPase?
Pumps H+ ions into organelles to maintain an acidic pH.
51
What are the three methods to disrupt H+ gradient created by V-ATPase?
1) Weak bases - ammonium chloride, chloroquine.
2) Ionophores - monensin (carrier) or channel formers.
3) V-ATPase inhibitors - bafilomycin.
52
What percentage of total endocytosis is non-clathrin in many cell types?
About 50%.
53
What are the three main types of non-clathrin endocytosis?
1. Macropinocyotsis
2. Phagocytosis
3. GEEC pathway
4. Caveolin-mediated endocytosis
54
What two types of non-clathrin endocytosis require actin but not dynamin?
Macropinocytosis and phagocytosis
55
What is involved in macropinocytosis and phagocytosis?
Formation of large endosomes and requires actin but not dynamin.
56
What is the GEEC pathway, and what does it involve?
1. Tubular endocytic vesicles
2. Uses lipid rafts, GPI-anchored proteins, and folate receptor
3. Does not use dynamin
57
What type of non-clathrin endocytosis requires dynamin?
Caveolin-mediated endocytosis
58
What are lipid rafts?
Lipid rafts are lipid microdomains enriched in cholesterol and sphingolipids.
59
Where are lipid rafts assembled?
Golgi Apparatus.
60
What do lipid rafts do?
1. Compartmentalize proteins.
2. Accommodate special transmembrane (TM) proteins, GPI-anchored proteins (GPI-APs)
3. House signalling molecules
61
What can caveoli do in relation to lipid rafts?
Caveoli can segregate lipid rafts under certain circumstances.
62
What endocytic mechanism specializes in internalizing lipid raft domains?
The GEEC pathway specializes in internalizing lipid raft domains.
63
What are caveolae?
Caveolae are small flask-shaped pits in the plasma membrane (roughly 70 nm in diameter), coated with caveolin (an integral membrane protein).
64
What makes caveolin unusual as a coat protein?
Caveolin is an integral membrane protein, unlike other coat proteins, and it forms a hairpin structure.
65
What is concentrated in caveolae?
Caveolae are cholesterol-rich and concentrate raft membranes. They can also concentrate signaling molecules.
66
What happens to endocytosed caveolae?
Endocytosed caveolae fuse to form caveosomes, which are neutral pH compartments distinct from other endosomes.
67
What role do caveolae play in transcytosis?
Caveolae play a role in transcytosis of albumin and other proteins across endothelia. This process is very rapid and distinct from “normal” transcytosis.
68
How do caveolae perform transcytosis of albumin?
Albumin binds to gp60 at the apical endothelium, activating transcytosis, which is dynamin-dependent.
69
What happens in caveolin knockout (KO) mice?
Caveolin KO mice are largely okay but have leaky capillaries and problems with their lungs, indicating a disruption in caveolae function.
70
How do caveolae function as signaling platforms?
Caveolae may function as signaling platforms by increasing the local concentration of signaling molecules, thereby enhancing the efficiency of signaling.
Caveolae can concentrate signaling molecules and act as signaling platforms. Signaling molecules bind to a conserved caveolin-scaffolding domain (CSD) on caveolin-1.
71
What is the role of caveolins in caveolae formation?
Caveolins are integral membrane proteins that deform the membrane to form the flask-shaped pits of caveolae.
72
What is the relationship between caveolae and lipid rafts?
Lipid rafts preferentially associate with caveolae, and caveolin-1 binds to cholesterol in the membrane.
73
What are the major components of caveolae?
The major component of caveolae is caveolin-1, and other proteins involved in their shape and function include cavins and Pacsin2.
74
What is the internalized compartment formed by caveolae?
The internalized compartment is called a caveosome, which is neutral pH and caveolin-1 positive.
75
What is macropinocytosis?
Nonspecific internalization process involving the uptake of fluid, particles, and membrane into the cell.
76
What is required for macropinocytosis to occur?
Macropinocytosis is actin-dependent and involves membrane projections, such as lamellipodia, circular ruffles, or blebs.
77
What is a macropinosome?
A macropinosome is an internalized vesicle with a diameter of >500 nm.
78
What happens to the cargo in macropinocytosis?
The cargo is eventually delivered to the lysosomes for degradation or processing.
79
What is the CLIC-GEEC Pathway?
1. Tubular-based, non-coated, clathrin and dynamin-independent form of endocytosis.
2. Occurs in the leading edge of migrating cells, involving polarized endocytosis.
80
What types of cargo are internalized through the CLIC-GEEC pathway?
The CLIC-GEEC pathway internalizes fluid, membrane, and lipid raft-associated GPI-anchored proteins.
81
What are the vesicles involved in the CLIC-GEEC pathway?
The internalized cargo is carried in CLICs (uncoated CI tubulovesicular carriers), which fuse to form GEECs (GPI-anchored protein-enriched early endosomal compartments).
82
What happens after the formation of GEECs?
After several minutes, GEECs fuse with normal early endosomes.
83
How do GEECs differ from early endosomes?
GEECs are more acidic than early endosomes.
84
What are GPI-anchored proteins?
GPI-anchored proteins are proteins anchored to lipid rafts, found mainly on the apical surface of polarized cells and have their sugar chain attachment at the ER.
85
Can you give an example of a GPI-anchored protein?
- Folate receptor
-> Binds folate at the cell surface and is internalized through the GEEC pathway.
86
What is the first step in the endocytosis of LDL?
The LDL particle binds to its receptor on the plasma membrane, initiating the internalization process.
87
How does LDL get internalized into the cell?
LDL is internalized via clathrin-dependent endocytosis, where the LDL receptor is recruited to clathrin-coated pits on the plasma membrane.
88
What happens to the LDL receptor and LDL particle once inside the cell?
In the early endosomes, the mildly acidic environment causes the LDL receptor to dissociate from the LDL particle. The LDL receptor is recycled back to the plasma membrane.
89
What happens to the LDL particle after dissociation from the receptor?
The LDL particle is delivered to the lysosome, where it is degraded into cholesterol, amino acids, and fatty acids.
90
How does the LDL receptor interact with clathrin-coated pits?
The LDL receptor has a domain that projects out and is recognized by the LDL receptor. This allows the receptor to be recruited to clathrin-coated pits, signaling its binding to adaptor protein ARH for endocytosis.
91
What occurs in the early endosomes?
The LDL binds to the LDL receptor at neutral pH (outside the cell), but the interaction is not stable in the mildly acidic environment of the early endosome. Here, the LDL is released from the receptor.
92
What happens to the LDL receptor after dissociating from the LDL particle?
The LDL receptor is segregated into tubules in the early endosome, detaches, and is recycled back to the plasma membrane to bind to another LDL particle.
93
What happens as the early endosome matures? (LDL Pathway)
The early endosome matures into a late endosome, and the LDL particle is transported into the lysosome for degradation.
94
What is the final fate of the LDL particle?
The LDL particle is degraded in the lysosome, releasing cholesterol, amino acids, and fatty acids, which are used by the cell.
95
What is the role of acidic pH in the process of LDL endocytosis?
The acidic pH in the early endosomes causes the LDL receptor to dissociate from the LDL particle, allowing the receptor to be recycled and the LDL to be transported to the lysosome for degradation.
96
What is the first step in the endocytosis of EGF and EGFR?
EGF binds to its receptor, EGFR, and this binding induces endocytosis.
97
What happens to the EGF-EGFR complex once internalized?
The EGF-EGFR complex remains stable in early endosomes.
98
How is EGFR inactivated after internalization?
EGFR is inactivated by sequestered into intraluminal vesicles within the early endosomes.
99
Why is it important for the EGF-EGFR pathway to be tightly regulated?
The pathway is crucial for cell division. If not properly regulated, excessive cell division can occur, leading to tumor formation, particularly in cancers like breast cancer.
100
What happens if the EGFR remains activated for too long?
If EGFR remains activated for too long, it can continuously signal for cell division, potentially leading to uncontrolled cell growth and tumor formation.
101
How does the EGF-EGFR pathway protect against excessive signaling?
After activation by EGF, the EGFR is internalized and inactivated, and lysosomes degrade the complex. This stops the signaling and makes the cell insensitive to further EGF signaling for a period of time, acting as a safety mechanism.
After EGF binding and internalization of the EGF-EGFR complex, the receptor is inactivated and degraded, preventing continuous signaling and excessive cell division.
EGFR inactivation helps prevent uncontrolled cell growth, as continuous activation of EGFR can lead to tumor formation, especially in cases like breast cancer.
102
What role does the lysosome play in the regulation of EGF-EGFR signaling?
The lysosome fuses with the early endosome, leading to the destruction of the EGF-EGFR complex and inactivation of the receptor, which ensures that the cell stops responding to EGF signals.
103
What happens when the EGFR is mutated, as seen in some cancers like breast cancer?
Mutations in EGFR can cause it to stay activated even without EGF binding, leading to continuous cell division and contributing to tumor formation.
104
What form of iron is found in blood plasma, and what does it bind to?
In blood plasma, Fe (III) (ferric iron) binds to the glycoprotein transferrin.
105
What are the two forms of transferrin?
1. apo-transferrin (iron-free)
2. Holo-transferrin (iron-bound).
106
How does transferrin deliver iron to cells?
Holo-transferrin binds to the transferrin receptor on the surface of reticulocytes, and the complex is internalized via clathrin-dependent endocytosis.
107
What happens to iron inside the early endosome?
In the early endosome, Fe(III) is released from transferrin, and it is reduced to Fe(II) by STEAP3. Then, Fe(II) is transported into the cytoplasm by the DMT1 transporter.
108
What happens to the transferrin-receptor complex after Fe(III) is released?
The apo-transferrin-receptor complex is recycled back to the plasma membrane, where apo-transferrin dissociates from the receptor due to the neutral pH at the cell surface.
109
What is the role of the transferrin receptor in endocytosis?
The transferrin receptor is a transmembrane protein that has a tyrosine-based motif allowing it to bind to AP2 and be internalized via clathrin-dependent endocytosis.
110
Why is iron important for cells?
Iron is essential for various cellular processes, including in mitochondria (for energy production), muscle cells (for myoglobin), and red blood cells (for hemoglobin).
111
What happens to transferrin when it is bound to iron at neutral pH?
When transferrin is bound to iron at neutral pH, it stays bound to the transferrin receptor on the cell surface.
112
What happens to transferrin and the transferrin receptor at the cell surface after the iron is released?
After iron is released in the early endosome, the transferrin-receptor complex is recycled back to the plasma membrane. At neutral pH, apo-transferrin dissociates from the receptor.
113
Why is it important that iron is tightly regulated and not freely available in the body?
The body does not have a lot of free iron floating around because iron can promote the growth of pathogenic bacteria. By binding to proteins like transferrin, the body controls iron availability and limits bacterial access.
114
What is transcytosis?
Transcytosis is a process that couples endocytosis and exocytosis, transporting cargo (such as immunoglobulins) from one side of a cell to another, typically in epithelial or endothelial cells.
115
What is the role of immunoglobulins in transcytosis?
Immunoglobulins like IgA and IgG are transported across epithelial or endothelial cells via specific receptors, allowing them to move from the basolateral to the apical side of the cell.
116
What are the receptors involved in IgA transcytosis?
pIgA (polymeric IgA) binds to a receptor on the basolateral side of epithelial cells. The receptor facilitates endocytosis of IgA and its transport to the apical side of the cell.
117
Where is IgA predominantly found in the body?
IgA is mainly found in mucosal tissues, including the respiratory and gastrointestinal tracts.
118
What happens to IgA after it binds to its receptor on the basolateral side of the epithelial cell?
1. pIgA binds to its receptor on the basolateral side of the epithelial cell and is endocytosed via clathrin-mediated endocytosis.
2. It then travels through several endosomes to reach the apical side of the cell.
119
What occurs at the apical side of the epithelial cell during IgA transcytosis?
At the apical side, pIgA is cleaved. The transmembrane (TM) portion of the complex remains in the cell, while IgA is released extracellularly into the lumen as sIgA (secretory IgA).
120
What is the structure of IgA and how does it differ from IgG?
IgA is similar to IgG in that both have heavy and light chains with antigen-binding sites, but IgA is a dimeric form with a piece between the molecules. This allows IgA to have four antigen-binding sites, making it effective at cross-linking antigens, such as viruses, and agglutinating them for phagocytosis.
121
Why is IgA important for mucosal immunity?
IgA is specialized for secretion and for crossing mucus membranes, such as in the intestines and respiratory tract. It helps neutralize pathogens by binding to them and preventing their entry into cells.
122
What happens after IgA aggregates pathogens like viruses?
After IgA binds to and aggregates pathogens (such as viruses), the immune system can recognize the complex and remove it via phagocytosis, followed by degradation in lysosomes.
123
How does the **transcytosis of IgA help prevent pathogen entry into the body?
By transcytosing across epithelial cells and being secreted into the lumen as sIgA, IgA neutralizes pathogens (such as viruses and bacteria) at mucosal surfaces, preventing them from entering the body.
124
What is the role of the Trans-Golgi Network (TGN) in the exit of cargo from the Golgi?
- Sorting station for outbound cargo, directing proteins to either the plasma membrane or endosomes.
- Acts as interface between the Golgi and the endocytic pathway.
125
What are the two main routes for TGN exit?
1. Secretion the the plasma membrane.
2. Transport to endosomes.
126
How can cargo transport from the TGN be classified?
1. Direct (TGN -> Target)
2. Indirect (through intermediates such as vesicles or tubular structures).
127
What is the difference between constitutive secretion and regulated secretion?
1. Constitutive
- continuous in all cells
- supplies the plasma membrane with newly synthesized lipids and proteins.
2. Regulated secretion
- only in specialized cells, where cargo (e.g., hormones, neurotransmitters) is stored in vesicles and only released in response to a signal.
128
Which type of secretion is found in specialized cells such as neurons and endocrine cells?
Regulated secretion, which involves the storage of cargo in vesicles that only fuse with the plasma membrane in response to a signal.
129
What is the process for packaging cargo for the regulated secretory pathway?
1. Cargo aggregates at the TGN.
2. Vesicles containing the cargo bud off from the TGN.
3. The vesicles undergo acidification, causing proteins to aggregate further.
4. The vesicles mature, and water transporters remove excess fluid.
5. The mature vesicle waits near the plasma membrane for a signal (often involving a Ca2+ influx) to trigger fusion with the membrane.
130
What is the key difference between synaptic vesicles and dense-core vesicles?
-> Synaptic vesicles contain small neurotransmitters.
-> Dense-core vesicles contain large neuropeptides and are found in neurons and endocrine cells. They undergo a maturation process involving aggregation and acidification.
131
How do dense-core vesicles mature?
1. Cargo in the vesicle aggregates at low pH in the TGN.
2. The vesicle acidifies further, causing protein aggregation.
3. The vesicle matures as it loses excess fluid and becomes concentrated with cargo.
4. The vesicle then waits near the plasma membrane for a signal, such as a Ca2+ influx, to trigger secretion.
132
What is the significance of tight junctions in polarized epithelial cells?
- Prevent lateral migration of transmembrane proteins, ensuring that apical and basolateral proteins are separated.
- Creates polarized domains, making it easier to study and target proteins to the correct membrane surface.
- maintain cell polarity.
133
How are proteins targeted to the apical or basolateral surface in polarized epithelial cells?
- proteins sorted at TGN based on signals recognized by receptors or carriers.
- sorting signals can be direct or indirect.
134
What are some common apical sorting signals in polarized epithelial cells?
1. O-linked glycosylation (occurs only in the Golgi)
2. N-linked glycosylation (begins in the ER and continues in the Golgi)
3. Protein motifs
4. GPI anchors, which associate with lipid rafts at the TGN.
135
How do tubular transport intermediates function in polarized epithelial cells?
- transport cargo from golgi to apical surface.
- use kinesins for movement.
- cargo likely selected based on its association with lipid rafts at the TGN.
136
What happens in a polarized epithelial cell when cargo is targeted to both the apical and basolateral surfaces?
- TGN can generate two population of vesicles
OR
- cargo may be dumped into one surface and later sorted via endocytosis and transcytosis to target correct membrane.
137
How is cargo packaged for the regulated secretory pathway?
- cargo aggregation at TGN
- vesicle containing cargo bud from TGN
- immature secretory vesicle undergoes membrane + lumenal content recycling
- secretory vesicle acidified
- mature secretory vesicle waits for signal at plasma membrane - signal causes Ca2+ influx.
138
What does 'apical' refer to in a polarized epithelial cell?
Apical side faces the lumen or external environment (e.g., the inside of the intestine or kidney tubule)
139
What does "basolateral" refer to in a polarized epithelial cell?
The basolateral side faces the blood supply or underlying tissue.
140
What is the function of tight junctions in a polarized epithelial cell?
Tight junctions prevent lateral migration of transmembrane (TM) proteins and stop the mixing of apical and basolateral constituents.
141
How are proteins sorted for apical or basolateral targeting?
Proteins have specific signals that are recognized by receptors or carriers at the trans-Golgi network (TGN).
142
What are the two main targeting routes for proteins in a polarized epithelial cell?
Direct (sorted at the TGN and sent straight to the correct membrane) and Indirect (sent first to one membrane, then transcytosed to the correct one).
143
What are the features of apical membrane secretory in polarized epithelial cells?
- Apical sorting signals are pleomorphic (varied in nature).
- O-linked glycosylation occurs only in the Golgi.
- N-linked glycosylation starts in the ER and continues in the Golgi; it is a weak apical signal that can be overridden by basolateral signals.
- Specific protein motifs help with sorting.
- GPI anchors assist in apical targeting by associating with lipid rafts at the TGN.
144
How do tubular transport intermediates deliver cargo to the apical membrane in polarized epithelial cells?
- transport occurs from the golgi to the apical cell surface.
- Kinesins move tubules along MTs.
- Cargo selectively likely involves partitioning into lipid rafts.
145
What are key features of basolateral membrane secretion in polarized epithelial cells?
- basolateral sorting signals are less complex than apical signals.
- signals include tyrosine-based motics and dileucine motifs in the cytoplasmic tail.
- clathrin is required for sorting most basolateral proteins.
- AP1B complex is the adaptor protein for basolateral targeting.
- the same cytoplasmic signals can be used to re-target proteins back to the basolateral membrane after endocytosis.
146
What is the difference between secretion in a polarized vs. non-polarized cell?
- polarized cells (e.g., epithelial cells) have distinct apical and basolateral membranes, requiring specific sorting mechanisms to direct proteins to the correct side.
- non-polarized cells (e.g., fibroblasts) do not have membrane segregation, so proteins are delivered randomly to different parts of the membrane.
- both cell types use the same secretory pathways, but in polarized cells, membrane-specific sorting proteins ensure targeted delivery.
- polarized cells need different proteins on their membranes to recognize and sort cargo appropriately.
147
what is the general structure of APs? (Adaptor Proteins)
- made of 4 domains, specific to type and tissue.
- can bind receptor tails via tyrosine and dileucine motifs.
- binds clathrin through ear.
- AP1 is involved in trafficking to cell surface and endosomes.
- AP3 is involved in trafficking to endosomes.
- AP4 on Golgi but function unclear.
148
What are the two pathways for transport from the Trans-Golgi Network (TGN) to lysosomes?
1. Direct pathway: TGN → Endosome → Lysosome
2. Indirect pathway: TGN → Plasma Membrane → Endocytosis → Lysosome
149
What is the function of clathrin in TGN-to-endosome transport?
Clathrin-coated vesicles help sort and transport proteins from the TGN into the endocytic pathway.
150
How do membrane proteins reach the lysosome?
1. Interact with COPII to leave the ER.
2. Reach the TGN, where they are sorted into the endocytic pathway using the AP3 clathrin adaptor.
151
How do soluble hydrolases reach the lysosome?
1. Bind to cargo receptors in the ER.
2. Travel to the TGN, where they interact with another cargo receptor for transport to the lysosome.
152
Why is N-glycosylation important for lysosomal membrane proteins?
It provides protection to the cell interior by stabilizing membrane proteins and preventing degradation.
153
What types of proteins are found in lysosomes?
1. Catabolic proteins (enzymes that break down macromolecules)
2. Activators (assist in enzyme function)
3. Soluble and membrane hydrolases (digest proteins, lipids, and carbohydrates)
4. Transporters and channels (regulate ion and metabolite movement in and out of lysosomes)
154
How are lysosomal membrane proteins transported and what ensures lysosomal stability?
1. Lysosomal membrane proteins rely on dileucine-based motifs (DXXLL or [DE]XXXL[LI]) and tyrosine-based motifs (YXXØ) for proper sorting.
2. These motifs interact with GGA or AP-3 adaptor complexes, which direct proteins from the TGN to endosomes and lysosomes.
3. LAMP-1 (Lysosomal-Associated Membrane Protein-1) helps maintain lysosomal stability and integrity, protecting the organelle from degradation.
155
Lysosomal Acid Hydrolases
- around 40 types
- function at low pH
- most need to be activated by proteolytic cleavage
156
What is the structure and function of GGAs?
- made of 4 domains
- able to bind receptor tail motif via VHS domain (M6PR and Sortillin)
- binds ARF via its GAT domain
- binds clathrin via hinge and terminal Ear domain
157
What are GGAs?
- Golgi-localized, gamma-ear-containing ARF-binding proteins.
- family of adaptor proteins involved in sorting of cargo proteins from the Golgi apparatus to endosomes and lysosomes.
- crucial role in clathrin-mediated vesicle formation, helping package cargo proteins into clathrin-coated vesicles for transport.
- have specific binding sites for sorting signals, such as dileucine motifs and interact with ARF proteins to faciltate vesicle budding from the Golgi.
158
Hydrolase Enzyme Synthesis & Targeting
- hydrolases (ex., cathepsins) enzymes synthesized in the ER.
- these enzymes are tagged with a specific signal in the Golgi apparatus to ensure they are directed to lysosome for their function.
- In the golgi, hydrolases are tagged with M6P, critical signal for lysosomal targeting.
- this tagging process occurs in two steps within the cis-compartment of the golgi.
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What are the two steps when tagging M6P to hydrolases in the cis-compartment of the golgi?
1. Addition of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to a mannose residue on the hydrolase.
(intermediate molecule with the GlcNAc attached to the mannose)
2. An enzyme called GlcNAc phosphoglycosidase removes the N-acetylglucosamine group, exposing the final mannose-6-phosphate (M6P) signal.
(this exposed M6P signal is the key marker that directs the hydrolase to the lysosome)
Different words:
1. At cis Golgi, GlcNacphosphotransferase transfers UDPGlcNAc to the N-linked
oligosaccharide attached to the
lysosomal hydrolase. The
mannose residue is in the N-linked
oligosaccharide
2. At medial Golgi, phosphodiester
alpha-GlcNAcase (*) removes
GluNAc to leave M6P.
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Why is the M6P signal important?
The M6P signal is recognized by the Mannose-6-Phosphate Receptor (M6PR) in the Golgi and endosomes, ensuring the hydrolase is properly transported to the lysosome.
The M6PR binds to the M6P-tagged hydrolases and facilitates their transport from the Golgi to the lysosomes.
Without proper M6P tagging, hydrolases won’t be correctly delivered to the lysosome, causing lysosomal storage disorders.
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What is the ICD disease? What is the impact?
- ICD (Inclusion Cell Disease), also known as mucolipidosis II, is caused by a mutation in the GlcNAc phosphotransferase enzyme.
- This mutation prevents the proper addition of the M6P tag to hydrolases, leading to the failure of enzymes to reach the lysosome.
- As a result, the hydrolases accumulate in the extracellular space and lysosomal storage issues occur, affecting cell function.
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What are the 5 groups that lysosomal storage disorders can be divided into?
1. Defects in glycan degradation
2. Defects in lipid degradation
3. Defects in protein degradation
4. Defects in lysosomal transporters
5. Defects in lysosomal trafficking
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Lysosomal storage disease
- characterized by enlarged lysosomes with excessive material (nervous system - particularly vulnerable to damage)
- individuals normally born health, with progressive development of symptoms.
- diseases are inherited in an autosomal-recessive fashion, except for X-linked recessive.
- speed and severity of symptoms depend on:
which cells are affected
the genetic background of individual
environmental factors
type of waste product accumulated
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Inclusion-cell disease (I-cell disease)
- most severe form of lysosome storage disease
- rare, inherited, recessive
- defect in transferring UDP-GlcNAc to hydrolases by N-GlcNAc - phosphotransferase (defect in hydrolases trafficking_
- most hydrolases absent from lysosomes
- severe-delayed development, short stature, coarse facial features, dyostosis multiplex, cardiomyopathy
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What are the general symptoms of I Cell Disease?
- Characterized by the accumulation of excess acid mucopolysaccharides in fibroblasts, connective tissue cells, and abnormal sphingolipids/glycolipids in lysosomes of visceral fibroblasts, macrophages, and nerve cells.
Key Symptoms:
- Mental retardation
- Skeletal changes (notably dysostosis multiplex)
- Dwarfism
- Deformed limbs
- Limited joint motion
- Spade-like hands
- Corneal clouding
- Hepatosplenomegaly
- Gargoyle-like facial features
Dysotosis Multiplex
- A form of mucopolysaccharidosis due to alpha-l-iduronidase deficiency.
- Results in abnormal accumulation of intracellular material.
- Excretion of dermatan sulfate and heparan sulfate in urine.
- Causes severe skeletal cartilage and bone development issues.
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What are the two types of M6P receptor?
1. CI M6P receptor
2. CD M6P receptor
- cytoplasmic tails contain sorting signals (dileucine and tyrosine signals)
- dileucine signal recognied by GGA.
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What is the structure of GGA (recognizes M6PR) ?
- 4 domains
- VHS: recognize DXXLL signals (binds to both forms of M6PR & to sortilin)
- GAT (GGA and TOM): binds to GTP bound Arf1.
- Hinge: binds to clathrin
- GAE (y-adaptin ear): binds to accessory proteins.
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Sortilin & GGA
- sortilin is a receptor that helps transport specific proteins, including propaposin (key activator protein)
- GGA adaptor proteins involved in sorting & transporting lysosomal enzymes - including those associated with Sortilin.
- Sortilin can bind directly to cargo proteins without requiring a M6P (mannose-6-phosphate) tag, unlike other receptors that rely on M6P for recognition.
- The C-terminus of Sortilin has motifs recognized by GGA proteins (specifically the DXXLL dileucine motif) and the AP-1 complex, which are crucial for sorting proteins to lysosomes.
- Sortilin binds to the C-terminus of prosaposin.
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Prosaposin & Saposins
- Prosaposin is a precursor protein that gets processed into saposins (A-D), which assist in the hydrolysis of sphingolipids.
- Saposins act as solubilizers or lipases, meaning they help break down sphingolipids into smaller components.
- Sphingolipids are important lipids in the membrane of cells, and their breakdown is essential for normal cellular function.
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What happens if GGA is truncated (lacking hinge and ear domains)?
1. Inability to interact with clathrin: The truncated GGA cannot bind to clathrin, disrupting clathrin-coated vesicle formation.
2. Impaired vesicle formation: Without clathrin recruitment, vesicle budding from the TGN is disrupted.
3. Disrupted cargo trafficking: The trafficking of lysosomal enzymes and other cargo to their correct destinations (like lysosomes) is affected.
4. Altered cargo recognition: While cargo binding may still occur, the lack of clathrin recruitment prevents proper sorting and efficient trafficking.
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Does targeting of membrane proteins to lysosome required GGA?
- no, it will still happen even if GGA is not functioning.
- this depends on clathrin.
- some membrane protein examples are: LEP, LAMPs, LIMPs.
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What is the function of AP-3?
- targets lysosomal membrane proteins such as LAMPs and LIMPs.
- LAMP-2 uses this mechanism.
Whereas Cathepsin B has be shown to use M6P receptor.
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Transport of most soluble hydrolases
It is generally done by the
mannose 6-phosphate receptor (M6P-Rc). The M6P-Rc uses GGA
adaptor proteins to enter into clathrin coated cargo vesicles.
174
Transport of activator proteins and some soluble hydrolases
Sortilin is another cargo receptor that uses GGA adaptor proteins. Sortilin
is involved in the transport of the activator protein prosaposin as well
as some soluble hydrolases that we didn’t discuss.
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Transport of lysosomal membrane proteins
LEP 100, LIMP 1-4 and LAMPs are
membrane glycoproteins. These group of proteins go to the lysosomes from the Golgi apparatus using adaptor proteins. The transport of lysosomal associated protein (LAMP) is mediated by the adaptor protein-3 (AP-3).
176
What is the retromer complex composed of?
1. Heterodimer sorting nexins (Snx1/2): induce and sense membrane curvature.
2. Heterotrimeric Vps35-29-26 (cargo-selective complex): cannot associate with membranes alone.
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How is Retromer recruited?
1. Binding to PI3P (via PX domain of Snx3).
2. Binding to Rab7 (via Vps35).
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What is the function of the Retromer?
- Returns mannose-6-phosphate receptor (M6PR) and sortilin from the late endosome to the Golgi.
- Clathrin-independent trafficking mechanism.
- Produces tubular buds with an unusual morphology.
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Phagocytosis
- specialized cells (macrophages, neutrophil, dendritic cells)
- cargo >500 nm diameter.
- actin dependent process (Rho GTPases) and receptor-dependent pathway: Ab on cargo binds to Fc receptor on phagocyte -> reorganization of actin cytoskeleton -> pseudopod formation
- pseudopods surround cargo in a zipper-life fashion
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What are the steps of phagosomal maturation?
1. phagosomes fuse with early endosomes, then late endosomes.
2. they acquire early endosomal proteins, then late endosomal proteins.
3. once sufficient LAMPs are acquired, phagosomes fuse with lysosomes.
4. this forms a degradative phagolysosome.
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How do some pathogens evade phagosomal maturation?
- convert the phagosome in an autophagosome or ER-like vesicle.
- convert the phagosome into a hybrid autophagosome-RAB7-positive phagosome.
- convert the phagosome into rough ER.
- inhibit phagosomal maturation by:
1. blocking at the RAB5-positive stage.
2. Preventing RAB7 function, keeping the phagosome in the RAB5-positive stage.
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Autophagy
- cargo acquired from within cell
- catabolic process for reusing components rather than de novo.
- non specific versus specific cargo: long lived proteins, protein aggregates, defective organelles.
183
What are the purposes of autophagy?
1. obtain nutrients under starvation conditions
2. turnover of defective organelles
3. removal of protein aggregates from cytoplasm
4. removal of bacteria/infectious agents
5. specialized purposes (e.g. liberation of cholesterol from lipid droplets)
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What are the three kinds of Autophagy?
1. Chaperone-mediated autophagy - KFERQ-tagged proteins bind to Hsc-70 and are translocated to lysosomes via LAMP2A.
- single cytoplasmic proteins containing by KFERQ pentapeptide sequence are recognized by and bind to Hsc-70
(heat shock cognate-70)à translocated to lysosome via the LAMP-2A
2. Microautophagy - proteins/Hsc-70 adhering to phosphatidylserine on the
surface of late endosomes are budded into internal vesicles.
- internalization of small regions of cytoplasm
3. Macroautophagy - large structures including protein aggregates and organelles are enclosed by a double-unit-membrane structure to form an autophagosome.
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Macroautophagy/autophagy
- turnover of bulk cytoplasm; damaged or unwanted organelles (mitochondria -> mitophagy, peroxisomes -> pexophagy)
- LC3 needed for phagophore expansion and closure.
186
What is LC3?
- microtubule-associated protein light chain 3.
- plays a key role in autophagy (helps form autophagosomes).
187
LC3-I vs LC3-II
1. LC3-I (soluble)
- initially produced as fully length LC3.
- C-terminal is cleaved.
- Binds to Atg3.
2. LC3-II (membrane bound - essential for autophagosome formation)
- LC3-I gets conjugated to PE.
- This process requires the Atg12-Atg5-Atg16L complex.
- LC3-II is now membrane-bound and helps form autophagosomes.
188
Steps of Macroautophagy
1. Phagophore membrane forms (origin may be ER or mitochondria)
2. LC3 processing
- LC3 cleaved
- Cleaved LC3 binds to PE -> becomes LC3-II.
- LC3-II inserts into the phagophore membrane.
3. Autophagosome formation
- The phagophore expands and engulfs cytoplasm or damaged organelles.
- targeted material is often ubiquitinated.
- P62 protein links ubiquitinated cargo to LC3-II for degradation.
4. The autophagosome fuses with endosomes, but LC3-II remains on the membrane.
5.
- The autophagosome fuses with lysosomes.
- Lysosomal enzymes degrade the contents for recycling.
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Maturation of autophagosomes
- autophagosomes merge with the endocytic pathway in a manner similar to phagosomes.
- first early-endosome like, then late-endosome like.
- they then fuse with lysosomes and the contents are digested.
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Rab Proteins
- Small GTPases (“on” when bound to GTP; “off” when bound to GDP)
- Bind “effector proteins” when in GTP state.
- Typical effector proteins include tethers, SNAREs, microtubule motors and lipid
modifying enzymes.
- Usually have one or more fatty acids at C-terminus that allows insertion in
membranes when in GTP state
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What family do Rab GTPases belong to?
- Ras superfamily; contains 5 main families.
- all members cycle between (active) GTP- and (inactive) GDP- bound forms: "molecular switches"
- Rab family make up the largest group
- Active, GTP-bound Rab proteins bind to effectors
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How do COPII vesicles leave the ER?
- COPII vesicles formed at ER exit sites are clustered together by the action of the golgin tethering factor p115.
- Rab1 recruits p115 on to these vesicles.
- These tethered vesicles then fuse in an NSF and SNARE dependent mechanism to form VTCs.
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Rab Cycle
- Rabs are brought to the membrane by GEFs (GTP exchange factors), different for
almost each Rab (so there are a lot of them!)
- Rabs in the GTP form (and on the membrane) can bind effectors (different Rabs
bind different effectors)
- Rab GAPs (there are many) inactivate Rabs by causing GTP hydrolysis. This
causes the loss of binding to effectors.
- Rab GDI removes Rab-GDP from membranes and sequesters it in the cytoplasm.
194
What are Rab Effectors?
- proteins that bind to Rabs only when the Rabs are in GTP state.
- one rab has many effectors that carry out different functions on the same organelle.
- many functions can be carried out by effectors:
tethering proteins (EEA1 for Rab5; p115 for Rab1)
regulators of motor proteins
regulators of lipid metabolism
195
How do Rab GTPases regulate endocytosis?
- lipid anchored, peripheral membrane proteins
- active Rab proteins bind to effectors to coordinate events on the vesicle (sorting, uncoating, tethering, fusion, locomotion)
- can serve as endosome identify markers
196
Where are Rab5 and Rab7 located?
1. Rab5 -> on early endosomes/phagosomes.
- early endosomes are abnormally large in cells overexpressing Rab5.
- Early endosomes are abnormally small in cells expressing a mutant
Rab5 that cannot bind GTP.
2. Rab7 -> on late endosomes/phagosomes, autophagosomes, lysosomes.
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Rab5
- master regulator of early endosomes
- activated Rab5 recruits effectors for tethering and fusion between early endosomes
- one key effector is PI 3-kinase, which converts PI into PI3P.
- PI3P important for early endosome identify, and helps recruit Rab5 effectors.
- Reinforces Rab5 activation, creating positive feedback loop for early endosome maturation.
198
Phosphoinositides in Endocytosis
- Phosphoinositides (PIs) are minor membrane lipids that act as trafficking signals.
- 7 different phosphoinositide species can be formed by phosphorylation at different positions on the inositol ring.
Each phosphoinositide has a specific subcellular location:
1. Early endosome → PI3P (Phosphatidylinositol 3-phosphate)
2. Late endosome → PI(3,5)P2 (Phosphatidylinositol 3,5-bisphosphate)
These phosphoinositides help recruit proteins that regulate vesicle trafficking, fusion, and maturation.
199
What effector binds to Rab5 and PI3P?
- EEA-1 (early endosome antigen 1)
- a filamentous, tethering factor
- assemble into homodimers
- binding to PI3P (FYVE domain), Rab5 (2 Rab binding domains)
- recruit SNARE components to Rab5 active domain
- EEA-1 directly interacts with SNAREs
200
Rab7
- master regulator of late endosomes
- requires 3 proteins for minus-end MT transport
1) RILP - links Rab7 to dynein-dynactin for transport
2) Dynein - motor protein that moves cargo toward the minus end of MTs (toward nucleus)
3) Dynactin - activated and enhances dyneins function - efficient transport.
201
ORP1L & Rab7 Minus-End Transport
- ORP1L = Cholesterol sensor on late endosomes (LE).
- When cholesterol is low → ORP1L loads βIII spectrin.
- βIII spectrin = Links late endosomes (LE) to dynactin.
- Dynactin = Recruits dynein, the motor that moves LE toward the minus-end of microtubules (toward the nucleus).
202
Rab7 for plus-end directed movement
- FYCO1, a Rab7 effector, competes with RILP binding
- FYCO1 connects LE/autophagosome to kinesin for plus-end
directed movement
- May occur in low cholesterol levels
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Rab7 & Vesicular Tethering
- Rab7 helps vesicle fusion by recruiting the HOPS complex.
- HOPS (Homotypic Fusion and Vacuole Protein Sorting) complex:
-> Allows fusion between late endosomes (LEs) (homotypic).
-> Allows fusion between late endosome (LE) & lysosome (L) (heterotypic).
- HOPS complex has 6 subunits.
- VPS33 binds to SNARE proteins (Vam3, Nyv1) to help vesicle fusion.
- VPS39 & VPS41 bind to Rab7 on vesicles to tether them together.
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how does Rab conversion happen during endosome maturation?
1. Rab5 GTP recruits Rab7 GEF
2. GEF catalyzes GDP -> GTP exchange on Rab7, activating it.
3. Activated Rab7 recruits TBC2 (a Rab5 GAP), which hydrolyzes Rab5 GTP to GDP.
4. GDP-bound Rab5 leaves the endosome membrane.
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How does Rab5 regulate its own removal?
1. Rab5 recruits a complex that brings Rab7 to the membrane.
2. This same complex removes Rabex5 (Rab5 GEF), stopping Rab5 activation.
3. Rab7 then recruits TBC2 (Rab5 GAP) to inactivate Rab5.
4. As a result, Rab5 is removed, converting the endosome to a late endosome.
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How does a late endosome (LE) fuse with a lysosome?
1. Rab7 is present on both LEs and lysosomes.
2. Proper SNARE proteins allow fusion.
3. The HOPS complex tethers the LE to the lysosome for fusion.
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What happens when a late endosome fuses with a lysosome?
- fusion creates a hybrid organelle called an endolysosome.
- endolysosomes have both LE and lysosome characteristics.
- as digestion occurs, the endolysoosme matures into a lysosome.
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What changes happen during endosome maturation?
- Movement in the cell (change in motors).
- Decrease in acidity.
- Change in lipid composition (PI3P → PI(3,5)P2).
- Closing off recycling pathways (retaining only degradative portions).
- Receiving lysosomal hydrolases from the trans-Golgi network (TGN).
- Switching fusion machinery (tethers and SNAREs).
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What are the two models of endosome-to-lysosome transition?
1. Model 1: Pre-existing compartment model → Late endosome fuses with a pre-existing lysosome.
2. Model 2: Maturation model → Late endosome gradually transforms into a lysosome.
