Endocytosis II Flashcards
1
Q
Pathway 1 + ex
A
Ldl
Atherosclerosis, heart attacks, stroke
2
Q
Pathway 2 + ex
A
Egf and it’s receptor
Growth factor, cancer
3
Q
Pathway 3 + ex
A
Iron - transferrin
4
Q
Pathway 4 + ex
A
Transcytosis
Igg (iga)
5
Q
Pathway 5 + ex
A
Endothelial transcytosis
Albumin,caveolae
6
Q
Pathway 6 + ex
A
Phagocytosis
7
Q
Pathway 1 = what happens
A
Ldl particle binds its receptor on pm
Internalized via clathrin dependent endocytsis
(Apolipoprotein b, large hdyrophobic domains, secreted by cells in liver, recognized by ldl r = can be recruited to clathrin coated pits = diff adaptor arh then clathrin pit internalized and fuse with endosomes)
(Similar for folate pathway and insulin)
8
Q
Pathway 1 = under what conditions
A
Ldl receptor complex in early endosomes and dissociates in mildly acidic environment
9
Q
Pathway 1 = what happpens to receptor
A
Returned back to pm = recycled
10
Q
Pathway 1 = what happens To ldl particle
A
Degraded in lysosome = release cholesterol, aas, fatty acids - bc endosome will fuse with lysosome
11
Q
Pathway2 = what does egf bind
A
Binds to receptor and induces endocytsosis
12
Q
Pathway2 = process
A
Internalized egf-egfr complexes are stable in early endosomes
Egfr inactivated by sequestration in intraluminal vesicles
13
Q
Pathway2 = whole process informally
A
Egfr sits on cell surface waiting for signal = can cause cells to divide- and if things go wrong = bc cell can divide too much = cancer
Requires strong egf signal = egf binds Egfr at cell surface = induces endocytsois —> receptor dimerizes —> internalized early endosome —> can be ub —> internalized early endosome —> segregated into internal vesicles —> late endosome —> internal vesicles have egf and Egfr = both degraded in lysosome, so safety mechanism so becomes insensitive
14
Q
Describe pathway 3 = types of iron
A
Fe ii (ferrous iron) vs fe iii (ferric iron)
Blood plasm fe travels as fe iii = binds to glycoprotein transferrin
15
Q
Describe pathway 3 = types of transferrin
A
Apo transferrin
Holo transferrin
16
Q
What happens to most plasma fe
A
Taken up by reticulocutes and loaded with transferrin r
17
Q
Describe process of pathway 3 = what is binding
A
Transferrin binds iron —> transferrin receptor
18
Q
Describe process of pathway 3 = internalization
A
2 holo transferrins bind to receptor on reticulocyte surface
Holo transferrin receptor complex internalized via clathrin dependent endocytsois
In early endosomes = feiii released from transferrin
Fe iii reduced to fe ii by steap3 = then transported back into cytoplasm by dmt1
19
Q
Describe process of pathway 3 = Recycling/end
A
Apo transferrin receptor complex recycled back to plasma membrane
Neutral ph causes Apo transferrin to dissociated from receptor
20
Q
Describe process of pathway 3 = In own words
A
Not much free iron in body - usually bound so harder for pathways to access it
Each transferrin binds 2 iron, binds transferrin receptor = tm protein tyrosine based motif = allows binding to ap2 and endocytosis via clathrin
At neutral ph
21
Q
What happens if transferrin has no iron attached
A
Transferrin gradually comes off receptor = may even reload
In early endosome = iron stripped from transferrin at acidic ph 6-6.5 = then iron transported
Transferrin receptor recycled with endosomes and go back to cell surface
22
Q
Describe pathway 4 - gen
A
Couples endocytsos and exocytosis
Transports cargo from one side of cell to another - epithelium, endothelium
Endocytsoed at Basolateral and exocytsoed at apical
23
Q
Describe pathway 4 - in detail
A
Immunoglobulins transported by receptors - secretory iga —> plgA, igg—> fcrn
Specialized for secretion and crossing membrane - specialized to neutral in bacteria, 4 binning sites = clumps them so macrophage can work, j chain of igg recognize by receptor on epi cells
24
Q
Where is iga
A
Mainly in mucosal tissue
25
Describe what plasma cells secrete
Dimeric iga
26
Describe plgA
Polymeric iga
At basolateal region of epithelial cells binds to iga
27
Describe transcytosis of iga
Complex clathrin dependent - endocytsoed, travels to apical side of cell
At apical side = plgA cleaved - tm still with cell, extracellualr with iga
SlgA released into luemn
(Iga bidns receptor on basolateral surface then brought by endosomes)
28
Describe exit from Golgi - gen
Tgn as sorting station for outbound cargo
Tgn as interface for Golgi and endocytic pathway
Too many diff destinations so form many diff types of transport intermediates
29
Describe exit from Golgi - direction of protein exit
Anterograde
30
Describe exit from Golgi -routes
2 main routes = secretion to pm
Cargo transport to endosome
Can be direct or indirect
Transport of cargo via carriers - vesicles or tubular intermediates
31
Name the 2 kinds of secretion
Constitutive vs regulated
32
Describe constitutive secretion
Occurs in all cells
Supplies pm with newly synthesized lipids and proteins - housekeeping
Immediate fusion with pm without signal
33
Describe regulated secretion
Some specialized cells
Cargo = hormones, neurotransmitters, digestive enzymes, pancreatic enzymes
Fusion with pm upon signal (liek calcium secretion etc)
34
Describe regulated secretion - types
Pathway variable = depends on cell types
Large uncoated vesicles - packed with proteins that aggregate
Aggregates pinched off in vesicles
Water channels, acidify more
MUST WAIT FOR SIGNAL
35
Describe dense core vesicles
DENSE core vesicles found in neurons and endocrine cells
Contain large neuropeptides
Rather than low molecular neurotransmitters
36
Describe packaging cargo for regulated secretory pathway - specifics
Cargo aggregation at tgn
Vesicle containing cargo bud from tgn
Immature secretory vesicle undergoes membrane + luminal content recycling
Secretory vesicle acidified = after buds, causes proteins to clump further and water transporter removes water = end up with mature secretory vesicle
Waits fo signal at pm - signal ca2+ influx can cause it to fuse with membrane
37
Describe image of packaging cargo for regulated secretory pathway
Clathrin coated retrieval vesicles = remove things that shouldn’t be there
Have some clathrin = allows recycling
But vesicles uncoated, not essential of sorting cargo, cluster at low ph in tgn
Uncoated = can be large
(Liek palade = pancreatic enzymes —> condensing vesicle —> zymogen granule)
38
Describe secretion in polarized cell = gen
Apical vs basolateral targeting
Regulated secretion in cells = towards apical surface
39
Describe secretion in polarized cell = types of targeting
Apical vs basolateral targeting
Can be direct or indirect
But need 2 pathways = diff surfaces with dif proteins
40
Describe secretion in polarized cell = what do tight junctions do
Prevent lateral Migration of tm proteins
Also prevent mixture of apical and basolateral constituents
41
Describe secretion in polarized cell = how are proteins sorted
Based on signals recognized by receptors/carriers at tgn
42
Describe apical sorting signals
Pleiomorphic
43
Name and describe apical sorting signals
O linked glycosyaltion = only occurs in Golgi
N linked glycosylatoon = begins in er and continues in Golgi- weak signal that can be overridden by basolateral targeting signals protein motifs
Gpi anchor
44
Describe gpi anchors - secretion to apical membrane
Normally associate with lipid rafts at tgn
45
Describe secretion to apical membrane = generally
Proteins sorted to Lipid raft domains
Proteins that are integral memrvame proteins with longe tm domains prefer raft domains
Proteins on inner least prefer raft domains
Raft domains tiny at surface but large at tgn
46
Describe tubular transport intermediates - pics
Made of raft domain
Required in pathway - secretion to apical membrane
Proteins going to apical surface bind kinesins - proteins move on my
Dynamin like proteins cut off tube and then move along mt
Pulling membrane = force servitude, naturally membrane forms tube
47
Describe when tubular transport intermediates
Golgi to apical surface in polarized epithelial cells
Independent of coat
Also happens in unpoalzired cells = will go but then mix with rest of cells
Also detach from early endosomes
48
Describe how tubular transport intermediates move
Tubules use kinesins to move
49
Describe mechanism of cargo selectivity for tubular transport intermediates
Probably involves partition of cargo into lipid rafts - no coats
50
Describe secretion to basolateral membrane - signals
Signals less complex
51
Describe secretion to basolateral membrane - name and describe signals
Protein that has =
Tyrosine based or dileucine based motifs embedded into cytoplasmic tail of basolateral proteins
52
Describe secretion to basolateral membrane - requires what
Requires clathrin = adaptor protein = ap1b complex - same signals ap2
(Sorted into ap1b vesicle at tgn, detaches from Trans Golgi and moves to basal surface mts in polarized cells, - in non polarized cells = signal efforts wasted bc will just all fuse together)
53
Describe secretion to basolateral membrane - describe use of cytoplasmic signals
Same cytoplasmic siganls can be used to retarget cargo back to basolatral membrane after its endocytsis
54
Describe general structure of aps
Adaptor proteins
4 domain, 2 small, 2 big specific to type and tissue
55
Describe general structure of aps= binds what
Binds receptor tails via tyrosine and dileucine motifs
Bind clathrin through ear
56
Describe general structure of aps= AP1
Involved in trafficking to cell surface - one subtype and also to endosomes
Ap1a = cooperates with gga, transferring intermediates in endosomes
Ap1b = targets cell surface
57
Describe general structure of ap = AP2
Endocytsis at celll surface
58
Describe general structure of ap = AP3
Also involved in trafficking to endosomes
Tgn to endosomes to endocytic pathway
59
Describe general structure of aPS = AP4
On Golgi
But function unclear
60
WHICH APS ON ENDOSOMES
Ap1a, ap3 and ap4
61
Name pathways for transport from tgn to endosomes
Direct = to endosome —> lysosome
Indirect = to pm —> endocytosis —> lysosome
62
transport from tgn to endosomes = what proteins needed
Catabolic proteins - activators, soluble and membrane hydrolases
63
transport from tgn to endosomes= protective mechanisms
Membrane proteins are n glycosylated to protect cell interior
64
transport from tgn to endosomes = transporter channels
Mutiple in lysosomeal mmerbmae
65
transport from tgn to endosomes= describe image/ex
Tm protein —> ap1a —> endocytic pathway - for some proteins
Need cargo receptor to get into endocytic pathway, no point where soluble hydrolases interac with coat protein
Proton pump
= LYSOSOME PACKED WITH PROETINS
MEMEbrane proteins that interact with cop2 - leave er —> tgn (ap3 clathrin adaptor)
Soluble hydrolases bind cargo receptor - already n glcyoslated —> tgn —> another cargo PROtein
66
Describe lysosomal membrane proTEINS
Transport replied on dileucine based motifs and tyrosine based motifs
Motifs interact with gga or ap3 complex
*get into endosome = need ap3 adaptor
67
Describe functions of lamps and limps
Lamp1 = lysosome stability and integrity
Lamp2 = similar to function lamp1, chaperone mediated autophagy
Limp1 = cell fusion
Limp 2 = endosome/lysosome biogenesis and maintenance, interact with fusion/fission machinery
68
Describe lysosomal acid hydrolases
40 types of acid hydrolases
Acidic hydrolases function in low ph
Most hydrolases need to be activated by proteolytic cleaveage
69
Describe structure ggas and name and explain all parts
4 domains
Diff adaptor
Sorting receptor = binds lysosomal enzymes
VHS = binds acidic cluster dileucine signal
Gat binds arf
Ear domain hinge domain = homologous to ap fam, single polypeptide
70
Describe specifically what parts of ggas bind what
Bind receptor tail motif vis vhs dom = sortilin and m6pr
Bidns arf via gat dom
Binds clathrin via hinge and terminal ear dom
71
Describe m6pr = tagging with m6p = details
Hydrolases, such as cathepsins, are synthesized in the ER and transferred to the Golgi where they are tagged in two steps with M6P.
1 = first the transfer of N-acetylglucosamine-1-phosphate from uridine disphosphate-N- acetylglucosamine to selected mannose residue.
2 = glcNac phosphoglycosidase removes N-acetylglucosamine to expose the M6Precognition signal.
In cis Golgi.
72
Describe m6pr = tagging with m6p = image
Binding of signal patch to recognition site - recognize by glcNac phosphotransferase enzyme
= mannoses phosphorualted = transfer glcNac p to mannose in catalytic site = blocks further mods by enzymes that modify n linked oligosaccharides = hydro lease stays in golgi
Tan - m6pr recognized phosphorylated mannose
73
Describe m6pr = tagging with m6p = image = specifically binding and releasing
m6p = binds receptor at ph6.5 in tgn , requires clathrin coat - ap1a and gag to sort mannose 6 phosphate receptor into clathrin pits of tgn —> gets to endosome = enzymes = come off at ph 5.5
At same time = other clathrin pits formed at diff cargos = targeted to basolateral surface etc= tgn forming diff transport intermediates at same time
Receptor recycling = retromer
74
Describe icd - image = what happens
Lysosomal hydrolase precursor not phosphorylated = no binding
Protein has no signal and randomly incorporated and goes to cell surface = end up with enzymes in blood and serum and not in lysosomes = diagnostic feature
Depends on degrees = if some residual activity = means less severe
75
Name lysosomal storage disorders - 5
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
76
Describe lysosomal storage disease = characterized by
Enlarged lysosomes with excessive material
Nervous system particularly vulnerable to damage - neurons, so mental retardation
77
Describe lysosomal storage disease = disease aspects
Individuals normally born healthy, with progressive development of symptoms
* Diseases are inherited in an autosomal- recessive fashion, except for X-linked recessive
78
Describe lysosomal storage disease = symomtpms
* Speed and severity of symptoms depend on: which cells affected, the genetic background of individual, environmental factors, type of waste product accumulated
79
Describe icd =disease characteristics
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
Big swollen luysosmes - most ppl die at young age if enzymes doesn’t work, less severe if mutation not as bad
80
I cell disease = protein/gene
UDP-N-acetylglucosamine N-acetylglucosaminyl-1- phosphotransferase (GlcNac Phosphotransferase)
81
I cell disease = common/severe mutation
There are ~40 known mutations. One causes a frame shift and another one a premature stop codon.
Truncates - enzyme not active so most severe
82
I cell disease = major symptoms
Severe -delayed development / short stature / coarse facial features / dysostosis multiplex* / cardiomyopathy
83
I cell disease = clincal diagnosis
High lysosomal enzyme activity in serum (gradually reaches cell surface) / Molecular genetic screening
84
Describe m6p signal addition = 2 steps formally
1. At cis Golgi, GlcNac-phosphotransferase transfers UDP- GlcNAc 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
85
Name the 2 types of m6p receptor
CI vs CD m6p receptor
86
Compare 2 types of m6p receptor
M6p receptors transport most hydrolases
Some overlap in cargo recognition between the 2 receptors
Cytoplasmic tails contain sorting signals - dileucine and tyrosine signals
87
What does adaptor gga recognize
Dileucine signal
88
Describe 4 domains of adaptor gga = VHS
Vps27, hrs, Stam =
Recognition of DXXLL signals (dileucine, acidic aa =x) binds broth forms of m6pr and to sortilin
89
Describe 4 domains of adaptor gga= GAT
Gga and Tom
Binds to gtp bound arf1 = small grapes, recruits cop1 to early googling, ap to tgn
90
Describe 4 domains of adaptor gga = HINGE
Binds to Clathrin - diff kinds clathrin proteins, diff vesicles formed with diff destinations
91
Describe 4 domains of adaptor gga = GAE
Gamma adaptin ear
Binds to acesssory proteins = modify how adaptor works
92
Describe sortilin
Another cargo receptor
Targeting protein (sortilin)
Prosaposin = transported by sortilin - cargo reaches lysosome, normal in pppl with icd
Prosaposin = translated in ear —> tgn then binds sortilin
Protease chops —> saposins = have diff functions - extract glycolipids, lipids stay in membranes and degraded inefficiently if no Prosaposin
93
Ex of disease is problem with sortilin
Gaucher’s disease
94
Gaucher’s disease = protein/gene
b-glucocerebrosidase / GBA - lipase missing
95
Gaucher’s disease = Common/severe mutations
N370S, 84GG, L444P
96
Gaucher’s disease = Major symptoms
Hepatosplenomegaly / anemia / skeletal disease / dementia / convulsions
97
Gaucher’s disease = Clinical diagnosis
Bone marrow aspirate / b-glucocerebrosiadase activity
Less severe than icd tho
98
Describe saposin function
Saposins b and c assist various lysosomal enzymes in catabolism of sphingolipid
- usually pulls out glycosulceramide
99
When does gauchers occurs
When issues with saposins c
100
What does metachromatic leukodystrophy occur
When arylsulfatase a and saposins b missing
101
Summarize sorting receptors for lysosomal proteins - mannose 6 phosphate receptor
Done by 46kda m6pr
M6pr uses gga adaptor proteins to enter into clathrin coated cargo vesicles
102
Summarize sorting receptors for lysosomal proteins - sortilin
Also sues gga adaptor proteins
Sortilin involved in transport of activator proteins Prosaposin as well as number of other activator proteins and hyrolases
103
How does sortilin recognize cargo
Does not require m6p tag for cargo recognition
Directly binds to cargo proteins
104
What does sortilin have at c terminus
Motifs recognized by gga - dileucine and gga and ap1a complex
= signals to bind
105
What does sortilin bind
C terminus of Prosaposin
106
Describe saposin a-d
For sphingolipid hydrolysis
Either as solubilizers or liftases
107
Describe exp - truncated gga
Take off hinge and ear domain and replace with gfp - so know which cells transferred
Dominant neg - express Protein = competes with endogenous protein and messes up pathway
Cos7 cell transferred with truncated gga -lacking hinge and ear domains to interact with clathrin
So then enzyme does not get into lysosome
108
Describe targeting of membrane proteins to lysosomes
LEP, LAMPs, LIMPs etc.
* Occurs normally even if GGA is not functioning. * Depends on clathrin
109
Describe ap3 function
Targets lysosomal membrane proteins such as lamps and limps
Recognizes dileucine motif on lamp2
Some also recognize tyrosine motifs, bc tyrosine aa aa hydrophobic aa
= ap3 likes proteins with short cytoplasmic domain and signal near membrane
110
Describe exp testing ap3
Use small interfering rna - 2 nts
Dsrna - cell will recognize as viral and chop up then when domain turned over = not replaced
No mu3
111
Transport of most soluble hyrolases
M6pr
- uses gga adaptor proteins to enter clathrin coated cargo vesicles
112
Transport of activator proteins and most soluble hyrolases
Sortilin is another cargo receptor that uses GGA adaptor proteins
Sortilin= involved in the transport of the activator protein prosaposin as well as some soluble hydrolases
113
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
transport of lysosomal associated protein (LAMP) is mediated by the adaptor protein-3 (AP-3).
114
Describe retromer = Gen
Coat wrapped around vesicle
2 parts = one involved in membrane curvature - curvature forms tubes
Retromer coat can recognize signal on cytoplasmic dom m6pr and sortilin = sorts tehse out of late endosome
115
What is retromer composed of
1. Heterodimer sorting nexins (Snx1/2): induce and sense membrane curvature. Snx3 has PX domain and associates with Snx1/2
2. Heterotrimeric Vps35-29-26: aka cargo selective complex. Cannot associate to membrane alone
116
What is retromer recruitment based on
1. Bind to PI3P (via PX domain of SN3)
2. Bind to Rab7 (via Vps35)
117
Does trafficking by retromer use clathrin
Trafficking by retromer = clathrin independent
118
What is retromer involved in
Return of m6pr and sortilin from late endosome to Golgi apparatus
Retromer = produces tubular buds with unusual mophologu
119
How are sortilin and mannose 6 phosphate receptor transported to and from Golgi - summary
Sortilin and m6pr = transported from golgi —> late endosome
In acidic ph of late endosome = lysosomal enzyme released from receptor
Then receptor is returned to Golgi via retromer coated vesicles
120
what does cathepsin use
M6pr
121
What does lamp2 use
Ap3 transport mechanism
