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MCB*2050 Exam > Lecture 11 > Flashcards

Lecture 11 Flashcards

1
Q

Lysosomes

A

plays key role in degradation of larger cellular components/organelles – autophagy

  • contains ~60 different soluble acid hydrolyase enzymes enzymatically active only at low pH (~4.6) of lysosome interior (lumen)
    resident lysosomal membrane proteins protected (‘shielded’) from degradation by attached lumen-facing carbohydrate groups (added to protein in ER and Golgi [N-glycosylation])
  • products of degradation are transported into cytoplasm – reused by various biosynthetic/ metabolic pathways
  • low pH in lysosomal lumen maintained by membrane-bound ATPase proton pumps pumps H+ from cytoplasm into lysosomal lumen
  • highly dynamic – lysosomes possess wide variety of shapes and sizes depending on organism/tissue/cell type
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2
Q

Biosynthetic pathway: trafficking proteins to lysosomes

A
  • lysosomal proteins (i.e., soluble and membrane proteins) synthesized and initially N-glycosylated in RER, then transported (via COPII vesicles) from ERES to Golgi (CGN)
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3
Q

in cis cisternae, glycosylated lysosomal-destined proteins are ___

A

further modified

i.e., phosphorylation of mannose residue(s) in protein’s core oligosaccharide(s) by N-acetylglucosamine phosphotransferase

  • M6P = signal ‘patch’ (lysosomal targeting signal)
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4
Q

proteins w/o M6P:

A

TGN → secretory vesicles/granules → pm/ECM

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5
Q

proteins with M6P:

A

TGN → clathrin-coated vesicle → late endosome → lysosome

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6
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes
* later portions of biosynthetic pathway intersect with ….

A

endocytic pathway at late endosomes

*responsible for uptake of plasma membrane proteins/receptors and extracellular materials/ligands (e.g, hormones, low-density lipoproteins, Fe3+, etc) and subsequent delivery to endosomes/lysosomes for internalization and/or degradation

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7
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

in TGN, soluble M6P-bearing lysosomal destined proteins (e.g., acid hydrolyases) recognized by

A

M6P receptor

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8
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

integral transmembrane protein:
lumenal-facing domain of M6P receptor binds to

A

M6P groups on soluble lysosomal-destined proteins in lumen of TGN

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9
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

M6P receptor mediates

A

subsequent concentration of soluble lysosomal (‘cargo’) proteins into nascent clathrin-coated transport vesicles

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10
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

cytoplasmic-facing domain of M6P receptor

A

binds to AP1 and GGA adaptor coat proteins

  • AP1/GGA mediate vesicle ‘cargo’ section (≈ Sec24 in COPII at ERES)
  • AP1/GGA proteins (also termed ‘AP complex’) also serve as ‘linker’ for clathrin-coat vesicle assembly
  • recruitment of AP1/GGA adaptor proteins from cytoplasm to TGN surface
    mediated by GTPase Arf1
    Arf1 ≈ Sar1 in COPII coat at ERES
    Arf1 also involved in initiation of COPI-vesicle assembly during retrograde transport from and within Golgi complex
  • Arf1 binding to GTP causes conformational change
    exposed lipid anchor in Arf1-GTP directs it from cytoplasm to outer leaflet of TGN membrane; initiates outward bending of membrane
  • AP1/GGA proteins recruited by Arf1-GTP also bind (link) to major component of vesicle bud ‘coat’…. clathrin
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11
Q

one molecule of clathrin consists of:

A

*three ‘light’ chain polypeptides & three ‘heavy’ chain polypeptides
* form three-legged structure: triskelion
* clathrin triskelions recruited from cytoplasm self-assemble to form outer ‘scaffolding’ (cage-like lattice) of ‘coat’ on growing vesicle

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12
Q

What does the inner layer of the ‘coat’ on growing vesicles consist?

A

inner layer of the coat consists of AP complex linked to Arf1 and M6P receptor bound to soluble lysosomal-destined ‘cargo’ proteins

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13
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

clathrin assembly promotes …

A

clathrin assembly promotes curvature (outward bending) of TGN membrane
* individual clathrin triskelions initially assemble to form hexagons that lie flat on membrane (cytoplasmic) surface
* triskelions subsequently self-assemble to form pentagons

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14
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes
* release (scission) of clathrin-coated vesicle from TGN membrane into cytosol mediated by…

A

mediated by dynamin - A large, soluble GTP-binding protein

  • dynamin recruited from cytosol to connection (‘stalk’) between growing clathrin-coated bud and TGN membrane
  • dynamin proteins assemble to form dynamin ring around stalk
  • GTP hydrolysis causes a conformational change in dynamin ring resulting in twisting and ‘pinching off’ (scission) of nascent vesicle
  • incubation of cell with gGTP (non-hydrolyzable analog of GTP) causes continued dynamin ring polymerization –
    results in long, extended ‘stalk’ and no scission of vesicle bud
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15
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes
* after ‘pinching off’ from TGN, nascent
vesicle’s clathrin coat disassembles
* here ARF1…

A

Arf1-GTP converted to Arf1-GDP

Arf1-GDP, AP1/GGA and clathrin triskelions released into cytoplasm and ‘recycled’ for additional rounds of clathrin-coat
assembly at TGN

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16
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

  • nascent vesicle with M6P-receptor bound soluble lysosomal ‘cargo’ proteins fuses with…
A
  • nascent vesicle with M6P-receptor bound soluble lysosomal ‘cargo’ proteins fuses with late endosome

vesicle trafficking/docking/fusion mediated by specific Rab/Rab effector & v/t-SNARES

at late endosome…
* acidic interior (~pH 5.0-5.5 in lumen) of late endosomes causes M6P receptors to dissociate from soluble lysosomal ‘cargo’ proteins (acid hydrolases)
TGN & TGN-derived vesicles = ~pH 6.5
* phosphate removed from M6P groups in soluble ‘cargo’ proteins

17
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

  • phosphate removed from M6P groups in soluble ‘cargo’ proteins prevents …
A

rebinding to M6P receptor

18
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

at late endosome…
* ‘empty’ M6P receptors ‘recycled’ back
to TGN via

A

Via retromer-coated vesicles

  • retromer complex ‘coat’ assembles on cytoplasmic surface of late endosome
    mediates membrane curvature and vesicle budding (how?) AND selects proper vesicle ‘cargo’ (i.e., ‘empty’ M6P receptors at late endosome)
  • retromer ‘coat’ disassembles after vesicle formation at late endosome
  • retromer vesicles (w/ various pm cargo and ‘empty’ M6P receptors) also traffic to plasma membrane
  • M6P receptor-cargo protein complexes at pm retrieved by receptor-mediated endocytosis and delivered back to
    late endosome
19
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

  • eventually, late endosome fuses with lysosome
A

late endosome trafficking and docking/fusion with lysosome mediated by organelle-specific
Rab/Rab effectors and SNARES

20
Q

Trafficking of soluble lysosomal proteins from TGN to lysosomes

eventually, late endosome fuses with lysosome

  • late endosome lumenal (soluble) contents released into …
A
  • late endosome lumenal (soluble) contents released into lysosome interior

soluble lysosomal ‘cargo’ proteins (i.e., acid hydrolyases) activated due to low pH (~4.6) of lysosomal lumen

21
Q

Trafficking of soluble lysosomal proteins from TGN to lysosome

  • fusion of late endosome with lysosome also results in…
A
  • lysosomal membrane proteins (‘cargo’) move laterally from late endosome membrane into lysosome membrane
    e.g., v-ATPase H+ pump - maintains low pH in lysosome interior

and…

  • materials from endocytic pathway also delivered to lysosome (via late endosome-
    lysosome fusion) for degradation
22
Q

Late endosomes: junction of biosynthetic and endocytic pathways

A

materials derived from plasma membrane and extracellular space via receptor- mediated endocytosis (endocytic pathway) delivered to endosome/lysosome for internalization/degradation

23
Q

Biosynthetic pathway:

A

Trafficking of nascent lysosomal membrane proteins from TGN to lysosome

24
Q

Endocytic pathway:

A

Internalization of pm proteins & extracellular materials/ligands delivered to lyso

25
Q

Summary:

Clathrin-coat vesicle assembly at TGN

*Arf1, AP1/GGA adaptors, and clathrin triskelions (‘lattice’) meidiate…

A

..membrane curvature and formation of budding vesicle

*AP1/GGA recognize and concentrate M6P receptors and their bound M6P signal patch-containing ‘cargo’ proteins (i.e., lysosomal soluble
*acid hydrolyases & lysosomal membrane proteins [e.g, V-ATPase H+ pump])
*vesicle scission (budding) mediated by dynamic & GTP hydrolysis
*clathrin coat disassembles and nascent vesicle traffics to and fuses with late endosome

26
Q

Which of the following is a GTP-binding ‘coat’ protein involved in the outward membrane curvature process during clathrin-coated vesicle formation at the trans-Golgi network?

a) Arf1
b) Ran
c) Sar1
d) ARC5
e) None of the above

A

a) Arf1

27
Q

_____ are ‘coat’ proteins that bind Arf1 and are responsible for concentrating soluble ‘cargo’ proteins in nascent clathrin-coated transport vesicles at the trans-Golgi network.

A

“Adaptor proteins” or “Clathrin adaptor proteins.”

These proteins play a crucial role in recruiting cargo molecules into nascent clathrin-coated vesicles at the trans-Golgi network (TGN) by binding both to cargo receptors and to clathrin via interactions with Arf1-GTP. This process ensures the concentration and sorting of specific cargo molecules into the forming vesicles for subsequent transport to their destinations within the cell.

MCB*2050 Exam (11 decks)

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