Lecture 11 Flashcards
Lysosomes
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
Biosynthetic pathway: trafficking proteins to lysosomes
- 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)
in cis cisternae, glycosylated lysosomal-destined proteins are ___
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)
proteins w/o M6P:
TGN → secretory vesicles/granules → pm/ECM
proteins with M6P:
TGN → clathrin-coated vesicle → late endosome → lysosome
Trafficking of soluble lysosomal proteins from TGN to lysosomes
* later portions of biosynthetic pathway intersect with ….
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
Trafficking of soluble lysosomal proteins from TGN to lysosomes
in TGN, soluble M6P-bearing lysosomal destined proteins (e.g., acid hydrolyases) recognized by
M6P receptor
Trafficking of soluble lysosomal proteins from TGN to lysosomes
integral transmembrane protein:
lumenal-facing domain of M6P receptor binds to
M6P groups on soluble lysosomal-destined proteins in lumen of TGN
Trafficking of soluble lysosomal proteins from TGN to lysosomes
M6P receptor mediates
subsequent concentration of soluble lysosomal (‘cargo’) proteins into nascent clathrin-coated transport vesicles
Trafficking of soluble lysosomal proteins from TGN to lysosomes
cytoplasmic-facing domain of M6P receptor
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
one molecule of clathrin consists of:
*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
What does the inner layer of the ‘coat’ on growing vesicles consist?
inner layer of the coat consists of AP complex linked to Arf1 and M6P receptor bound to soluble lysosomal-destined ‘cargo’ proteins
Trafficking of soluble lysosomal proteins from TGN to lysosomes
clathrin assembly promotes …
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
Trafficking of soluble lysosomal proteins from TGN to lysosomes
* release (scission) of clathrin-coated vesicle from TGN membrane into cytosol mediated by…
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
Trafficking of soluble lysosomal proteins from TGN to lysosomes
* after ‘pinching off’ from TGN, nascent
vesicle’s clathrin coat disassembles
* here ARF1…
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
Trafficking of soluble lysosomal proteins from TGN to lysosomes
- nascent vesicle with M6P-receptor bound soluble lysosomal ‘cargo’ proteins fuses with…
- 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
Trafficking of soluble lysosomal proteins from TGN to lysosomes
- phosphate removed from M6P groups in soluble ‘cargo’ proteins prevents …
rebinding to M6P receptor
Trafficking of soluble lysosomal proteins from TGN to lysosomes
at late endosome…
* ‘empty’ M6P receptors ‘recycled’ back
to TGN via
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
Trafficking of soluble lysosomal proteins from TGN to lysosomes
- eventually, late endosome fuses with lysosome
late endosome trafficking and docking/fusion with lysosome mediated by organelle-specific
Rab/Rab effectors and SNARES
Trafficking of soluble lysosomal proteins from TGN to lysosomes
eventually, late endosome fuses with lysosome
- late endosome lumenal (soluble) contents released into …
- 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
Trafficking of soluble lysosomal proteins from TGN to lysosome
- fusion of late endosome with lysosome also results in…
- 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
Late endosomes: junction of biosynthetic and endocytic pathways
materials derived from plasma membrane and extracellular space via receptor- mediated endocytosis (endocytic pathway) delivered to endosome/lysosome for internalization/degradation
Biosynthetic pathway:
Trafficking of nascent lysosomal membrane proteins from TGN to lysosome
Endocytic pathway:
Internalization of pm proteins & extracellular materials/ligands delivered to lyso
