Lysosome

Question 1

What is the lysosome?

Answer 1

‘Digestive’ organelle – degrades all types of macromolecules (lipids, sugars, nucleic acids, and proteins, etc.)
—-> Low-density lipoproteins via receptor-mediate endocytosis, damage pm receptors, etc.

Question 2

What does the lysosome play a key role in?

Answer 2

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

Question 3

What are some facts about the lysosome?

Answer 3

Contains approx. 60 different soluble acid hydrolase enzymes
Enzymatically active only at low pH (4.6) of lysosome interior (lumen)
—-> Only turn on at lysosome
Resident lysosomal membrane proteins are protected (‘shielded’) from degradation by attached lumen-facing carbohydrate (oligosaccharide) groups (attached to the protein in ER and Golgi (N-glycosylation))
Products of degradation are transported into the cytoplasm – reused by various biosynthetic/metabolic pathways
Low pH in lysosomal lumen maintained by membrane-bound ATPase proton pumps
Pumps H+ from the cytoplasm into the lysosomal lumen
Lysosomes are highly dynamic – possess a wide variety of shapes and sizes depending on organism/tissue/cell type

Question 4

What does the biosynthetic pathway do?

Answer 4

Trafficking proteins to lysosomes

Question 5

What happens in the biosynthetic pathway?

Answer 5

Lysosomal proteins (i.e., soluble and membrane proteins) are synthesized and initially N-glycosylated in RER, then transported (via COPII vesicles) from ERES to Golgi (GCN)

Question 6

What happens in the cis cisternae of the biosynthetic pathway?

Answer 6

In the cis cisternae, glycosylated lysosomal destined proteins are further modified
Phosphorylation of mannose residue(s) in protein’s core oligosaccharide(s) by N-acetylglucosamine phosphotransferase

Question 7

Explain what the M6P does?

Answer 7

M6P = lysosomal signal ‘patch’ (targeting signal)
Proteins without M6P: TGN –> secretory vesicles/granules –> pm/ECM
Proteins with M6P: TGN –> clathrin coated vesicles –> late endosome –> lysosome

Question 8

How does the trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 8

Later portions of the biosynthetic pathway intersect with the endocytic pathway
—-> Responsible for the 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

Question 9

What happens in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 9

In TGN, soluble M6P- bearing lysosomal destined proteins (acid hydrolases) are recognized by M6P receptor
Integral transmembrane protein
The luminal-facing domain of the M6P receptor binds to M6P groups on soluble lysosomal-destined proteins in the lumen of TGN

Question 10

What is the first step in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 10

1) M6P receptor mediates the subsequent concentration of soluble lysosomal (‘cargo’) proteins into nascent clathrin-coated transport vesicles
Membrane ‘cargo’ receptor proteins at ERES involved in binding soluble proteins to be packaged and transported into COPII-coat vesicles
The cytoplasmic-facing domain of the M6P receptor binds to AP1 and GGA adaptor coat proteins
Cytoplasmic proteins with multiple protein-protein-binding domains
AP1/GGA = Sec24 in COPII at ERES mediate vesicle ‘cargo’ section
AP1/GGA proteins (also termed ‘AP complex’) also serve as ‘linker’ during clathrin-coat vesicle assembly
Recruitment of AP1/GGA adaptor proteins from the cytoplasm to TGN surface mediated by Arf1
Arf1 = Sar1 in COPII at ERES is a GTPase (regulatory) protein
Arf1 is also involved in the initiation of COPI-coated vesicle assembly during retrograde transport within and from the Golgi complex
Arf1 binding to GTP causes a conformational change
Exposed lipid anchor in Arf1-GTP recruited from cytoplasm to outer leaflet of TGN membrane
AP1/GGA proteins recruited by Arf1-GTP also bind (link) to a major component of vesicle bud ‘coat’… clathrin

Question 11

What is the second step in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 11

2) After ‘pinching off’ from TGN, the nascent vesicle’s clathrin coat disassembles
Arf1-GTP converted to Arf1-GDP
2a) Arf1-GDP, AP1/GGA, and clathrin triskelions released into the cytoplasm and ‘recycled’ for additional rounds of clathrin-coat assembly at TGN

Question 12

What is clathrin?

Answer 12

One molecule of clathrin consists of three ‘light’ chain polypeptides and three ‘heavy’ chain polypeptides
Form three-legged structure: triskelion
Clathrin triskelions recruited from the cytoplasm self-assemble to form outer ‘scaffolding’ (cage like lattice/outer cage) of ‘coat’ on growing vesicle
The inner layer of the coat consists of AP1/GGA proteins linked to Arf1 and M6P receptor bound to soluble lysosomal ‘cargo’ proteins
Clathrin = sec13/sec31 outer scaffolding in COPII vesicle formation at ERES
Clathrin assembly promotes curvature (outward bending) of TGN membrane
Individual clathrin triskelions initially assemble to form hexagons that lie flat on the membrane (cytoplasmic) surface
Triskelions subsequently self-assemble to form polygons
Transition to hexagons and pentagons serves as a mechanical driving force for membrane curvature

Question 13

How is clathrin related to the trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 13

Release of the clathrin-coated vesicle from TGN membrane mediated by dynamin
Large, soluble GTP-binding protein
Dynamin recruited from cytoplasm to connection (‘stalk’) between growing clathrin-coated bud and TGN membrane
Dynamin proteins assemble to form a dynamin ring around the stalk
GTP hydrolysis causes a conformational change in dynamin ring resulting in twisting and ‘pinching off’ (scission) of nascent vesicle
•Incubation of cell with gamma GTP (non-hydrolyzable analog of GTP) causes continued dynamin ring polymerization- results in long, extended ‘stalk; and no scission of vesicle bud

Question 14

What is the third step in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 14

3)Nascent vesicle with M6P-bound soluble lysosomal ‘cargo’ proteins fuses with the late endosome
Vesicle trafficking/docking/fusion mediated by specific Rab/Rab effector & v/t-SNARES
ERES- to- Golgi COPII vesicle trafficking

Question 15

What happens in the late endosome of the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes in relation to the late endosome?

Answer 15

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 insoluble ‘cargo’ proteins
Prevents rebinding to M6P receptor

Question 16

What is the fourth step in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 16

4a) ‘empty’ M6P receptors ‘recycled’ back to TGN via retromers
Vesicles responsible for trafficking ‘back’ (retrograde) from late endosome to TGN
Retromer vesicles formed by retromer complex
More recently discovered protein ‘coat’ = (COPI/II & clathrin)
Retromer complex ‘coat’ assembles on late endosomes (cytoplasmic) surface
Mediates membrane curvature, vesicle budding and selects proper vesicle ‘cargo’ (i.e., ‘empty’ M6P receptors)
This occurs at the late endosome

Question 17

What is the fifth step in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 17

Retromer vesicles (w/M6P receptors) also traffic to the plasma membrane
Capture soluble (and membrane) lysosomal proteins that ‘escaped’ from TGN via the secretory pathway

Question 18

What is the sixth to eighth steps in the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 18

6-8) M6P receptor-cargo protein complexes at pm retrieved by receptor-mediated

Question 19

What continues after the steps of the TGN of trafficking of soluble lysosomal proteins from the TGN to lysosomes?

Answer 19

Eventually, mature late endosome fuses with a lysosome
Late endosome trafficking and docking/fusion with lysosome mediated by organelle-specific Rab/Rab effectors and SNAREs
Late endosome luminal contents released into lysosome interior
Soluble lysosomal ‘cargo’ proteins (i.e., acid hydrolyses) activated due to low pH (4.6) of lysosomal lumen
The fusion of mature late endosome with lysosome also results in…
Nascent lysosomal membrane proteins move laterally from the late endosome to the lysosome membrane
e.g., v-ATPase H+ pump – maintains low pH in lysosome interior AND
Materials from the endocytic pathway

Question 20

What are the late endosomes?

Answer 20

Junction of biosynthetic and endocytic pathways 
Materials derived from the plasma membrane and extracellular space via receptor-mediated endocytosis (endocytic pathway) delivered to endosome/lysosome for internalization/degradation
Extracellular ligands (low-density lipoproteins, hormones, Fe3+, etc.) or damaged pm receptors
Involves early endosomes, multivesicular body (late endosome), and ESCRT machinery.