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BIOC 212 > Traffic > Flashcards

Traffic Flashcards

1
Q

What act as a timer for folding of proteins in the secretory pathway?

A

ER N-linked glycosylation → addition and removal of glucoses and mannose

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2
Q

Which coated transport vesicles are responsible for anterograde and retrograde?

A

Anterograde → COP-II → vesicle transport from ER to Golgi

Retrograde → COP-I → vesicle transport fro Golgi back to ER (because of mistakes from COP-II)

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3
Q

Where does Clathrin-coated vesicle transport to?

A

Transport from Golgi and Plasma membrane → endosomes (or lysosomes)

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4
Q

What are the 4 general steps common to all vesicle formation (COP-I, COP-II, CCV)

A
  1. Initiation → some event on the membrane
  2. Coat formation:
    - Cytosolic adaptor poteins interact with initiator (in the membrane)
    - Adaptors collect TM cargo, or cargo receptor
    - Coat = protein framework is formed on top of adaptors to shape the vesicle bud from the membrane
    (Summary = initiator, adaptor, coat)
  3. Fission → bud is pinched off to separate vesicle from membrane
  4. Uncoating → coat is removed to allow vesicle targeting and fusion (to know where to go)
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5
Q

What is the main difference between Ras GTPase and Rab GTPase?

A

Rasis a sub-family of Rab = signal transduction → GTP-bound state
Rab = vesicle targeting → binding sites for various effectors (not specific)

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6
Q

What are GAP and GEF?

A

GAP = GTPase Activating proteins → stimulate GTP hydrolysis → conformational change

GEF = Guanin Exchange Factors → cause release of GDP and binding of GTP

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7
Q

What are the roles Sar1 and Arf?

A

*Both Ras GTPase proteins (monomeric switches)
Sar1 → COP-II vesicle initiator

Arf → COP-I vesicle initiator

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8
Q

What is the process of COP-II vesicle initiation?

A

Forms at specific ER exit sites:
- proteins with exit signals collected (cargo receptors)
- misfolded proteins are kept away (calnexin)

TM GEF at exit site → induces GTP binding by Sar1-GDP → conformational change exposes amphipatic helix (exposed in Sar1-GTP state) → partially inserts into membrane to initiate vesicle formation
All on cytosolic side

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9
Q

What are Sec23, Sec24, Sec13 and Sec31?

A

Sec23 and Sec24 = Adaptors (inner coat)
Sec23 recognizes Sar1
Sec24 recognizes cargo receptor/cargo TM protein

Sec13 and Sec31 = Coat proteins → bind adaptors and shape the membrane into a vesicle (outer coat)

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10
Q

What does the COP-II coat formation step involve?
(Where does the energy for coat formation come from?)

A
  1. Adaptor proteins: Sec23 → Sar1 and Sec24 → TM cargo proteins/ cargo receptors for lumenal protein (If protein is in the lumen, receptor has to be TM to interact with Sec24)
  2. Coat proteins (Sec13/31) bind adaptors and shape the membrane into a vesicle
  3. Completed coat pinches the vesicle off from membrane
    *Energy for shaping + pinching off the vesicle is only from protein interactions, not from GTP hydrolysis
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11
Q

What is the process of the COP-II uncoating step of vesicle formation?

A

Adaptor (Sec23/24) acts as GAP → hydrolyse Sar1-GTP to Sar1-GDP:
- GTP hydrolysis (slow) = timer
- Sar1-GDP releases from vesicle membrane (amphipatic alpha helix hidden back)
- adaptors separate from Sar1 and coat separates from adaptors

*Uncoating is necessary for vesicle fusion at Golgi

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12
Q

Why do we say ER exit is a bulk flow?

A
  • proteins in the ER transported to Golgi and PM by default with no exit signal
  • Many proteins exported much more efficiently, whil some others return to the ER

→ ER resident proteins are transported to Golgi by bulk flow, but have a signals that return them to the ER

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13
Q

What are the ER exit signals?

A

TM proteins have exit signals on cytosolic side:
- di-phenylalanine (FF) at C-terminus (type 1)
- Asp-X-Glu (DxE) with a sequence
- recognized by Sec23/24 adaptors

Lumenal proteins are recognied by various cargo receptors = TM proteins with exit signals (FF in C-terminus facing cytosol)

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14
Q

What are the different ER exit signals?

A
  • FF at C-terminus (type 1 TM proteins have C-terminus in cytosol)
  • Asp-X-Glu (DxE) within a sequence

Lumenal protein bind to TM receptors with FF signal

*recognized by Sec23/24 adaptor

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15
Q

What are the ER retrieval signals for lumenal proteins?

A
  • KDEL-COO- at C-terminus → recognized by TM KDEL receptor which itself has a KKxx motif at the cytosolic C-terminus
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16
Q

What is the ER retrieval signal for TM proteins?
(retrieval = wants to get back from Golgi → ER)

A
  • KKxx-COO- at cytosolic C-terminus (type 1 TM proteins)
  • NH3+ - MxxRR at cytosolic N-terminus (type 2 TM proteins)
    (proteins different directionalities so different motifs)

*Motifs are recognized by the COP-I coat adaptors

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17
Q

What is the initiator in COP-I coated vesicle formation?

A

Arf1-GTP → amphipatic helix into membrane (Ras GTP + Arf-GEF)

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18
Q

What is the function of adaptors in COP coated vesicles?

A
  1. bind activated Sar1/Arf
  2. Bind TM cargo protein/cargo receptors
  3. Act as GAP for the initiator to dissociate the coat (change conformation of the initiators to hide the amphipatic alpha helix)
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19
Q

What are the adaptor and Coat proteins for the COP-I coated vesicle?

A

Large multi-subunit complex:
Adaptors → beta/sigma, gamma/zeta subunits → collect cargo
COP-I coat → alpha/beta’ subunits assemble on adaptors, shape and pinch off vesicle from membrane

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20
Q

What starting point/end point transport does COP-II transport ensure?

A

Anterograde
ER → Golgi
Golgi → Early endosomes, Late endosomes, Secretory vesicles, extracellular space
Early endosomes → late endosomes → Lysosomes
Secretory vesicles → extracellular space

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21
Q

What starting/end point transport does COP-I transport ensure?

A

Golgi → ER
Early endosomes → recycling endosomes → extracellular space or direclty to extracellular space
Late endosomes → Golgi
Secretory vesicles → Golgi
*Not from late → early endosomes

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22
Q

What starting/end point does Clathrin-coated vesicles transport ensure?

A

Early endosomes → Late endosomes → lysosomes
Extracellular space → early endosomes

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23
Q

Which molecule is the initiator of Clathrin-coated vesicle formation?

A

PI-phosphate in PM and Golgi → 2 fatty acid tails + glycerol + phosphate + sugar with -OH

PI can be phosphorylated by PI kinases → PI-phosphates provide binding sites for different proteins:
- PI(4, 5)P2 recognized by PM clathrin adaptors → dynamin
- PI(4)P recognized by Golgi clathrin adaptors

*Any other phosphorylation combination activates another signaling cascade, but not the formation of CCV

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24
Q

What are the different CCV adaptors and coat?
What are their roles in the formation of CCV vesicles?

A

AP-1, AP-2, other bind to PI-phosphate (in their specific combination) + bind cargo in membrane:
- many different signals for selection of cargo (mono-Ub, phosphorylation, etc.)
- Arf GTPase assists some adaptors, but does not initiate CCVs

Clathrin coat binds the adaptors

*Can also have actin involved in coating?

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25
Q

In what conformation does Clathrin coat CCV?

A

Many Clathrin triskelions → oligomers, 3 heavy chains + 3 light chains
Assemble on adaptors to shape the membrane and form “coated pits”
Clathrin forms a cage around vesicle:
- cannot pinch off vesicles by itself → need Dynamin (main difference between CCV and COP-I/II)

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26
Q

How does CCV fission of the vesicle from the membrane occur?

A

Dynamin = Rab GTPase → pinches off CCV
(NOT a Ras)

Dynamin monmers assemble in GTP-bound state → oligomeric ring at base of bud → GTP hydrolysis causes coordineated constriction of ring that pinches off the vesicle → fission → Dynamin ring disassembles in the GDP-bound state

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27
Q

What is auxilin?

A

DNAJ → clathrin-binding domain + J domain

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28
Q

How does Clathrin uncoating occur?

A

Pi-phosphates modify PI(4,5)P2 → weaken adaptor binding
Auxilin (DNAJ) binds to assemble clathrin cage + activates HSC70

HSC70 binds clathrin → induces conformational change → disassemble coat into triskelions → Clathrin release from HSC70 and recyclesto membrane
*Need help fo chaperones for uncoating

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29
Q

What are the new and old model of perception of how the Golgi apparatus work?

A

Old model → vesicle transport cargo between layers
New model → each layer matures and becomes the next layer:
- Golgi-resident proteins are carried backwards by COP-I
- Clathrin-coated vesicles carry cargo to PM and to endosomes

Cytosolic protein matric maintains organization of the stack → cis, medial, trans

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30
Q

What is Dynamin? What is its role?

A
  1. Dynamin = Rab GTPase, monomer → assemble in GTP-bound state → oligomeric rigns at base of bud
  2. GTP hydrolysis → coordinated constriction of ring → pinches off the vesicle (CCV FISSION)
  3. Dynamin ring disassemble in GDP-bound state
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31
Q

What are the 2 mechanisms that ensure that vesicles transport their contents to the correct acceptor membrane?

A
  1. Rab GTPase proteins → provide specificity of vesicle targeting and attachement to acceptor membrane (which subway to get on and which stop to get out at)
  2. SNARE fusion proteins → specificity during fusion of vesicle with acceptor membrane
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32
Q

Why are the multiple steps in the secretory pathway important before the vesicle gets to its final destination? (ex: stop by the Golgi apparatus)

A

1 Add PTM that provide function and specificity to the protein
2. If mistakes are made during direct transport, can’t go back, multiple steps allow for backwards mechanisms too

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33
Q

How is the Golgi organized?

A

Organized in stacj of membranes:
- Cis-Golgi (close to ER)
- medial-Golgi
- Trans-Golgi (closer to the plasma membrane)

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34
Q

How are the N-linked glycans modified in the Golgi?

A
  1. Removal of mannose
  2. Addition of different sugars, often with negative charges
35
Q

What is O-linked glycosylation?

A

PTM in the Golgi → complex oligossacharides are attached to Ser and Thr side chains
- Compared to N-linked glycans, O-linked glycosylation does not add the same glycans to every proteins

36
Q

What are the functions of glycosylation in the Golgi?

A
  1. Promote protein folding:
    - Makes folding intermediates more soluble (prevent aggregation)
    - Sequential modifications → Glyco-code → Progression folding or degradation
  2. Sugars have limited flexibility → protects from proteases, stabilizes protein structure (protective coat)
  3. Signaling hubs (regulation of development) by -OH groups on sugars
37
Q

What is the role of proprotein converses in the Golgi?

A
  1. Some PM and extracellular proteins made as long, inactive form at the ER → cur by proprotein convertases into shorter, active form at the Golgi
  • Proteases recognize specific patterns of AA
  • Cleavage often activates proteins by removing inhibitory region
38
Q

Give an example of Proprotein convertases and its effect.

A

Proinsulin = inactive polypeptide
- In proinsulin, N-term and C-term are close by disulfide bonding
- Convertases remove the middle section → 2 remaining sections for active form
- Prevents premature signaling by insulin at the ER

39
Q

Give an example of regulation by proteases in the Golgi for AFT6.

A

AFT6 is activated by converts proteolysis in the Golgi

Regulation is by trafficking:
- BiP covers ER exit signal on AFT6 → when more unfolded proteins → less Bip to cover the exit signal → AFT6 brought to the Golgi → proteases only in Golgi → Cleavage of AFT6 TM protein → cytosolic part goes in the cytosole to act as a TF for UPR

40
Q

What are the Rab function in vesicle traffic?

A

Rab-GTP proteins provide specificity of vesicle targeting and attachement to acceptor membrane

Rabs can act at several steps in vesicle targeting:
- Vesicle budding: assist cargo selection + coat formation
- Connect vesicle to motors on cytoskeleton for transport
- Tether vesicles to acceptor membrane → specificity
- Recruit SNARE fusion protein

41
Q

What are the specific roles of Rab1, 2, 3, 4, 5, 7, 9, 11?

A

Rab1 = ER → Golgi
Rab2 = Golgi → ER
Rab3 = exocytosis of secretory vesicles (out of cell)
Rab4/11 = recycling from endosome → PM
Rab5 = Endocytosis from PM → endosome
Rab7/9 = Golgi and early to late endosomes

42
Q

What are the characteristics/mechanisms of the Rab membrane anchor?

A

Rabs have 2 prenyl lipid groups attached at their C-termini

In GDP-bound state → Lipid covered up by other proteins (GDI) → Rab = soluble, not associated with the membrane
In GTP-bound state → lipid modification exposed → anchor Rab to the membrane → Rab-effector proteins become attached to the membrane through Rab-GTP

43
Q

How is Rab activated? What is the effect of this activation?

A

Specific GEF on membrane → anchored, active Rab-GTP (GEF is linked to formation of vesicle coat)

Rab-GTP works through effector proteins:
- Attach vesicle to motor proteins
- Tether vesicle to target membrane
- Activate PI kinases and GEFs to make more Rab-GTP in clusters on acceptor membranes

44
Q

What is the Vesicle Rab Cycle?

A
  1. Vesicle Rab is activated by GEF on donor membrane (starting point of transport) → packages into vesicles:
    - Some interact with cargo
    - Assist uncoating
    - Attach to motors (vesicles don’t fuse, they travel on cytoskeleton)
  2. Vesicle Rab-GTP binds specifically tether on the acceptor membrane
  3. After fusion, GAPs on target membrane inactivate Rab
  4. Inactive vesicle Rab-GDP is recycled through cytosol to donor membrane (bound to GDI)
45
Q

What are the structures of the cytoskeleton?

A

Protein filaments = Actin filaments → shorter, often clustered at PM, highly crosslinked into network
Microtubules = Tubulin → longer, thicker, organized around centrosome near nucleus

Both connected to each other and anchored to organelles and PM (cortical cytoskeleton)

46
Q

What are the motor proteins responsible for vesicle transport in the cell along the cytoskeleton?
What level of specificty do they provide?

A
  1. Myosins on actin
  2. Dyneins (AAA proteins) and kinesins on microtubules
    *Not same mechanisms and families, but all ATP-dependent

No targeting specificity, but bring the vesicle to general location of acceptor membrane

47
Q

What is the first determinant of vesicle targeting specificity?

A

Tethers
Many Rab effectors (Not actual Rab) = tethers → long proteins which connect vesicle with acceptor
- Bound to specific vesicle Rab on one end, and a specific membrane site on the other end (Rab in somes cases, not all)

48
Q

What are different types of tethers? (Name and Location)

A

Tethers of different sturctural families act at different organelles

1st Type: Coiled-coil tethers → 2 alpha-helices → recognition signal for the Rab within the arms → within the Golgi and on endosomes (helps to keep structure of the Golgi apparatus)

2nd Type: Multisubunit tethers act at other compartments:
- ER ↔ Golgi
- Golgi → PM
- Endosomes and lysosomes

49
Q

What are the characteristics of Coiled-coil tethers in the Golgi?

A

Dimers with coiled-coil structures → remain assembled after use

Form Golgi matrix:
- Maintain organization of the Golgi stack
- connect vesicles → Golgi
- Anchored to membrane, or attached by GTPases
- Long filaments with multiple Rab binding sites

*Have Rab-GTP binding sites
Coiled-coils always stay in the Golgi matrix, not recycled

50
Q

What are 3 different multisubunit tether families?
(tether = Rab effectors)

A
  1. ER → Golgi + within Golgi: TRAPPI, II
  2. Endosomoses → lysosomes: HOPS/CORVET
  3. ER and PM: CATCHR family (exocyst)
51
Q

How do the TRAPPI multisubunit tethers function?

A

TRAPPI work with p115 (coiled-coil tether)

  1. TRAPP1 acts as GEF for Rab1 on Golgi (brings Rab-GTP to attach to the membrane)
  2. Rab1 binds coiled-coil tether (bound to membrane by Rab1)
  3. CC tether (p115) binds to vesicle (induces conformational change in CC tether) and hands vesicle to recognition domain of TRAPPI (closer to the membrane)
  4. TRAPPI helps organize SNARE which will help the vesicle fuse to the membrane

*3 domains → Vesicle recognition, GEF, SNARE recognition

52
Q

How does the exocyst work for at the plasma membrane?

A

It is a member of the CATCHR family of multisubunit tethers

  1. Before tethering but after uncoatin, some subunits are on either the vesicle or the PM
  2. subunits on vesicles and PM interact to for a complete 8-mer complex tether → bring the vesicle close to the PM
  3. May bring SNARE to membrane to start fusion
53
Q

What are the different types of endosome tethers? And their characteristics?

A

To endosmoses → Rab5 (on the vesicle) interacts CORVET (tether) (early endosome → late endosome)
To lysosomes → Rab7 (on the vesicle) interacts HOPS (lat endosome → lysosome)
*HOPS and CORVET act as bridges between vesicle and target

  • Complexes have the same core subunits
  • Different end subunits bind different Rabs
  • Also bind SNARES
54
Q

What does the endocytosis pathway involve?

A

*Transport of bacteria and virus
- Vesicle traffic between PM, early endosome and trans-Golgi
- Early endosome matures → multivesicular body (MVB) and late endosome → Membrane swtich from Rab5 → Rab7
- late endosomes matures → lysosomes
- Other vesicles traffis to lysosomes

55
Q

Explain the Rab cascade that occurs in the maturation of the early endosome to late endosomes in the endocytosis pathway.

A

Endosome has GEF for Rab5 →
Rab5-GTP effectors:
*Rab5-GTP has exposed prenyl group that anchors it to the membrane
- CORVET (tether) → GEF for Rab7 → Rab7-GTP

Rab7-GTP effectors:
- GAP for Rab5 → Rab5-GDP → inactive (hidden prenyl groups → dissociates from the membrane)
- recruits more Rab7

As Rab5 vesicle fuse with early endosomes, more and more Rab7 is activated, less and less Rab5 stays active → membrane becomes late endosomes

56
Q

What is specific of the vesicle landing sites in early endosomes?

A

NOT different in thickness, just in composition:
- phosphorylated PI → provide additional binding sites for vesicle tethers
- Rab5 + effector proteins
- GEF activity → more Rab5-GTP
- Has CORVET tethers

57
Q

What is the general function of SNAREs and what are the 2 classes of SNAREs?

A

Membrane proteins that carry out vesicle fusion
- Rabs and tethers can recruit SNARES to fusion sites

v-SNARE on vesicles recognize t-SNAREs on target membrane:
- Complexes form after tethering
- unique combinations of v-SNAREs and t-SNAREs determine targeting specificity

58
Q

What are the characterstics of v-SNAREs and t-SNAREs?

A

v-SNAREs: monomers with single TM helical domain
t-SNAREs: trimers → 1TM + 2 proteins that interact with the TM protein

Specific v- and t-SNAREs form stable tetramers → mutliple SNARE complexes form at target site t induce vesicle fusion
*Unique combination of v- and t-SNAREs

59
Q

How does SNARE folding occur?

A
  1. v-SNARE monomer is not stably folded
  2. t-SNARE trimer is partially stable 3-helix bundle
  3. v- and t-SNARE fold into very stable 4-helix bundle
  4. Folding process pull the membranes closer together:
    - physical strain like a spring

After fusion, SNARE complex = unstrained, stable, inactive

60
Q

Is SNARE folding ATP dependent? GTP dependent?

A

NOPE → spontaneous process

61
Q

How does fusion of a vesicle occurs?

A
  1. SNARE complexes form in a ring around the vesicle contact site
  2. SNARE TM anchors are bent and strained → force that holds the membranes together
  3. Outer and inner layers of the membrane fuse
  4. Strain in the SNARE compelx is releived (SNARE protein are not bent anymore
62
Q

How does SNARE dissociation occur (after vesicle fusion to the receptor membrane)?

A

After fusion → SNARE complex is stable, unstrained and inactive

AAA-family ATPase (NSF) (+ accessory proteins) dissociates v- t-SNAREs → essential for continuation of vesicle traffic
→ t-SNAREs become active again
→ v-SNAREs recycled back to their donor membrane by vesicles

63
Q

How does NSF interact with SNARE? What is its role?

A

A member of the AAA ATPase family responsible for dissociation of v- and t-SNAREs
- NSF-ATP binds SNARE complex through adaptator proteins (alpha-SNAP)
- NSF twists and pulls during ATP hydrolysis
- Multiple cycles of ATPase unwind the SNARE helices

64
Q

Which vesicles mediate endocytosis from PM → early endosomes?

A

CCV (always when leaving PM)

65
Q

How does receptor recycling occur? (To bring them back to where they have their function after they have transported a molecule to its target destination)

A

Empty receptors (in the early endosomes) are recycled back → PM

If need to be degraded
FINISH or DELETE

66
Q

Where are extracellular ligands brought to when entering the cells?

A
  1. Interact with TM PM receptor → transported to early endosomes for sorting
  2. pH of early endosomes = lower than extracellular space (6.0) → changes average charges on proteins, weaker interaction → ligands separate from TM receptor
  3. Free ligans progress to lysosomes
67
Q

How is cholesterol transported from the outside → inside of the cell?

A

*LDL = low density, lipoprotein, cholesterol carrier

  1. Cholesterol = hydrophobic so protected by a carrier extracellularly (LDL)
  2. LDL-Receptor in PM recognizes the LDL
  3. CCV formation → coating → uncoating → fusion to early endosome
  4. Receptor → recycled back to the membrane, Cholesterol + carrier → fused to lysosome
  5. Carrier is digested into monomers and released with cholesterol for cell to re-use
68
Q

What is the importance/role retrograde traffic? What molecules does it involve?

A

Endosomes → PM or trans-Golgi:
- extracellular receptors → PM
- receptors that bring proteins to the lysosomes → returned to Golgi

Involves membrane tubules/tubular vesicles → not round coated vesicles
Requires the Retromer protein complex
*Has nothing to do with COP-I, COP-II, CCV

69
Q

What happens when TM receptors have to be degraded?

A
  1. marked for endocytosis by modification with mono-Ub at PM: (Mono-Ub receptor still transports cargo to early endosomes and then this happens)
    - not Poly-Ub
    - recognition by CCV adaptors
    - if Ub is removed → recycled to PM
    - If Ub not removed → signal for lysosomal degradation
  2. Early endosomes mature into MVB (multivesicular bodies) (then transports cargo to early endosomes and then this happens) → invagination and pinching-off membrane → MVB contents can’t be recycled to PM anymore
    (only early endosomes contents can be recycled)
  3. MVB fused with lysosomes that has necessary components/enzymes for degradation
70
Q

How does MVB formation occur?

A

MVB invagination → secreis of ESCRT proteincomplexes shape and pinch off vesicles into the lumen of an endosome

  1. ESCRT-0 binds PI(3)P (on the cytosolic side of the vesicle) → collects mono-Ub cargo protein
  2. ESCRT-I and ESCRT-II form the neck around the bud
  3. ESCRT-III forms oligomers to pinch off the bud → form the vesicle
71
Q

What is different for TM protein degradation vs non-TM proteins?

A

Need ESCRT → to make MVB because it’s the only way to isolate the cytoplasmic side of a TM so it doesn’t start signaling cascades (which we don’t want because this protein is ready for degradation)

72
Q

What does the inside of a lysosome look like?
How is the environment kept acidic?

A

pH ~ 50
Has difference acid hydrolases:
- nucleases, proteases, glycosidases, phosphatases, lipases, sulfatages, phospholipases, H+
-H+ come in through the H+ pump → ATP-dependent

73
Q

How is a lysosome formed?

A

MVB fuse with vesicles containing proteases and other enzymes to become lysosomes

74
Q

What are the names of the 4 pathways for lysosome degradation?

A
  1. Endocytosis
  2. Autography
  3. Phagocytosis
  4. Macropinocytosis
75
Q

What is Autophagy?

A

It is a pathway for lysosomes degradation → for Large-scale digestion of cytosole and membranes since doesn’t have the size/unfolding limit of proteasome
- degrades with specificity
ex: mitochondria

Upregulated during starvation to release free amino acids

Lipids and proteins are transported through cytosol to phagophore vesicles → grows to enclose contentes in an autophagosomes (double membrane, large range of sizes) → Autophagosomes fuse with lysosome → autolysosomes → to digest contents

76
Q

What is the difference between a phagophore and an autophagosome?

A

The phagophore = vesicle that elongates to go around the content that is targeted form autophagy → becomes the autophagosome when it is a double membrane enclosed vesicle (double membrane because both side where the membrane of the phagophore

77
Q

What is a concrete example of vesicle fusion and recycling?

A

fusion of vesicle to neuronal terminals when release of neurotransmitters

78
Q

Describe the process of formation of synaptic vesicles in a nerve cell

A
  1. Delivery of synaptic vesicle membrane components to presynaptic PM
  2. OPTION A) Endocytosis of synaptic vesicle membrane components to form a new synaptic vesicle direclty → Budding of synaptic vesicle from endosome
    OPTION B) Endocytosis of a synaptic vesicle membrane components and delivery to endosomes
    *Endocytosis by CCV
  3. Loading of neurotransmitter into the synaptic vesicle
  4. Secretion of neurotransmitter by exocytosis in response to AP
79
Q

What is homotypic fusion?

A

When in some fusion events, donor and target membranes are the same:
- Fusion of COP-II vesicles into vesicular-tubular cluster that becomes cis-Golgi
- re-formation of ER and Golgi after cell division

Both membrane have identical v- and t-SNAREs, already in complexes and inactive
* 2 vesicles each have a t-SNARE and a v-SNARE and t- interacts with v- of other vesicle → on larger vesicle (cis-Golgi)

SNAREs must be separated by NSF to allow new fusion

80
Q

How are lysosomes recycled after having degraded the contents from the late endosome?

A
  1. Degrades the contents
  2. Contents exit the vesicle by pores
  3. Only enzymes left in the lysosomes → ready to fuse with another late endosomes to degrade its contents
81
Q

What are the general steps of retromer formation?

A

Same steps as vesicle formation even if they are very different

  1. Initiation
  2. Coat formation
  3. Fission
  4. Uncoating
82
Q

What are retromers?
Explain the formation steps in details

A
  1. rab5-GTP and Rab7-GTP initiates retromer formation
    2a. Cargo adaptor (Vps26/29/35) binds Rab and selects transmembrane cargo protein
    2b. Sorting nexin SNX complex binds adaptor and PI(3)P (on the membrane) → causes membrane to curve by interacting with lipids (not by forming a rigid coat)
    *Different SNX proteins for Golgi and PM traffic
    2c. SNX + adaptor form a complete retromer unit

Tubule formation:
2d. Clusters of retromer shape the membrane into a long tube (not a rigid cage like COP-I/II or CCV would do)

  1. Dynamin homologs and cytoskeletal motor protesin pinch off the membrane
  2. GTP hydrolysis by Rab cause dissociation or retromer complex and uncoating → necessary for fusion with target
83
Q

What is a retromer?
What is a complete retromer unit?

A

Retromer = a complex of proteins that has been shown to be important in recycling transmembrane receptors, important in retrograde traffic, from endosomes → PM or trans-Golgi
SNA (sorting nexin) + adaptor (Vps26/29/35)
- Same step as coated vesicles for formation
- Form tubules for transport

84
Q

What are the steps of PTM in different layers of the Golgi?

A
  1. cis Golgi: SORTING + phosphorylation of oligosaccharide on lysosomal proteins
  2. cis cisterna: removal of Mannose
  3. medial cisterna: removal of Mannose + addition of GlcNAc (N-acetylglucosamine)
    1st residue added
  4. trans cisterna: addition of Gal + addition of NANA
  5. trans Golgi: sulfation of tyrosines and carbs + SORTING → lysosome/PM/secretory vesicle

BIOC 212 (15 decks)

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