endosomes and lysosomes

Question 1

what is a lysosome

Answer 1

• digestive organelle that degrades macromolecules and cellular organelles/components
• plant and yeast cells have a large central lysosome called a vacuole

Question 2

features of lysosomes

Answer 2

• contains soluble acid hydrolyase enzymes which are active in its low pH (~ 4.5)
• resident lysosomal membrane proteins are protected from degradation by sugars attached to their luminal domains
• products of degradation are transported to the cytosol
• low pH is maintained by a membrane-bound proton ATPase pump

Question 3

What targets proteins to the lysosome

Answer 3

• phosphorylation of a mannose residue in the proteins core oligosaccharide
• produces M6P which is the lysosomal sorting signal

Question 4

structure of clathrin

Answer 4

• one molecule has 3 light chain polypeptides and 3 heavy chain polypeptides - forms a triskelion structure
• clathrin triskelions assemble to form the outer scaffolding on the growing vesicle
• individual clathrin triskelions initially assemble to form hexagons
• then begin to form pentagons which is the driving force for membrane curvature

Question 5

clathrin-coated vesicles

Answer 5

• clathrin triskelions assemble to form the outer lattice of the coat of the growing vesicle
• the inner layer of the coat consists of the AP complex
• clathrin lattice assembly promotes curvature of the membrane

Question 6

Clathrin-coated vesicle budding at the TGN

Answer 6

• release of the vesicle from the TGN membrane is mediated by dynamin (a GTP binding protein)
• dynamin is recruited to the stalk between the growing clathrin-coated bud and the TGN membrane
• dynamin polymerize to form a dynamin ring around the stalk
• GTP hydrolysis causes twisting and pinching off of the vesicle

Question 7

what do GTPys do in clathrin-coated vesicle synthesis

Answer 7

• cause dynamin ring polymerization to continue which results in extended bud ‘stalk’ and no scission occurs

Question 8

trafficking proteins to the lysosome: step 1

Answer 8

• in the TGN, soluble M6P-bearing lysosomal proteins are recognized by the M6P receptor
• M6P receptor mediates the subsequent concentration of lysosomal proteins into nascent clathrin-coated vesicles
• cytoplasmic portion of the M6P receptor recruits the AP complex to the surface of the vesicle and in turn, the AP complex recruits clathrin triskelions to form the outer coat of the vesicle

Question 9

trafficking proteins to the lysosome: step 2

Answer 9

• after the clathrin-coated vesicle buds off the trans golgi the clathrin coat and AP complexes depolymerize and can be reused in another round of vesicle formation

Question 10

trafficking proteins to the lysosome: step 3

Answer 10

• the uncoated vesicle now transports to and fuses with the late endosome. The M6P receptor releases its cargo into the lumen of the late endosome

Question 11

late endosome

Answer 11

• has an acidic interior (~ pH 5) which causes M6P receptors to dissociate from soluble lysosomal cargo proteins

Question 12

trafficking proteins to the lysosome: step 4 and 5

Answer 12

• free M6P receptors are recycled
• vesicles carrying free M6P receptors can return to the TGN or fuse with the PM delivering M6P receptors to the cell surface

Question 13

trafficking proteins to the lysosome: steps 6-8

Answer 13

• phosphorylated lysosomal enzymes can be sorted at the trans-Golgi to the cell surface and secreted

Question 14

trafficking proteins to the lysosome: step 9

Answer 14

eventually, the mature late endosome fuses with the lysosome
- late endosome luminal contents are released into the lysosome interior
- soluble lysosomal cargo proteins are activated due to low pH of the lysosome

Question 15

Multivesicular body

Answer 15

• late endosome fragments
• MVB intraluminal vesicles contain materials derived from the PM
• MVB fuses with the lysosome
• MVB membrane proteins move laterally into lysosome membrane
• MVB soluble proteins are released into the lumen

Question 16

endocytosis

Answer 16

• opposite direction of secretory pathways
• materials move into cells via vesicles formed at the PM
  2 different forms…
  1. bulk-phase endocytosis
  2. receptor-mediated endocytosos

Question 17

receptor-mediated endocytosis

Answer 17

• responsible for concentrating and internalizing specific extracellular materials bound to specific receptors on the PM
• involves clathrin-coated vesicles
• brings in PM proteins destined for degradation

Question 18

phagocytosis

Answer 18

• uptake of large, particulate materials from the extracellular space of specialized cells
• e.g. ingestion of microbes by macrophages

Question 19

what makes general endocytosis different from phagocytosis

Answer 19

selective internalization of PM components
- e.g. cell surface PM receptors bound to extracellular ligands

Question 20

steps in receptor-mediated endocyctosis

Answer 20

1. receptor becomes activated by binding to a specific, soluble extracellular ligand
2. receptor-ligand complex diffuses laterally in the PM and accumulates in coated pits
3. clathrin-coated vesicle pinches off from the PM via dynamin and the clathrin coat disassembles
4. vesicle transport from the PM to early endosome and vesicle fusion is mediated by the cytoskeleton, Rabs and v/t-SNAREs
5. the early endosome becomes the late endosome via activation of an ATP-dependent proton pump that acidifies the lumen
6. the late endosome then partitions itself into recycling and sorting compartments

Question 21

coated pits

Answer 21

specialized regions of the PM where receptors concentrate and molecules needed for clathrin-coated vesicle formation reside (such as clathrin and AP2)

Question 22

what is the early endosome

Answer 22

responsible for sorting and recycling extra-cellular materials via endocytosis
- more acidic interior than endocytic vesicle causes dissociation of the transmembrane receptor from its soluble ligands

Question 23

how does the early endosome mature into the late endosome

Answer 23

• via activation of an ATP dependent proton pump that acidifies the lumen
• the late endosome then partitions itself into recycling and sorting compartments

Question 24

recycling compartments of the late endosome

Answer 24

detaches as vesicles from the rest of the organelle and traffics back to the plasma membrane
- recycled receptors at the PM are used fro additional rounds of receptor-mediated endocytosis

Question 25

the pH of endocytic vesicles

Answer 25

pH = 7
- extracellular space is generally neutral, hence the lumen of endocytic vesicles is also neutral

Question 26

the pH of early endosomes

Answer 26

pH = 6
- slightly more acidic than the lumen of incoming vesicles
- lower pH causes ligands to fall off their receptor

Question 27

the pH of late endosomes

Answer 27

pH = 5
- once the early endosome has received its cargo from the PM it activates a proton pump that pumps protons into the lumen in an ATP dependent manner
- lumen becomes acidified, turning it to the late endosome