Advanced Cell biology - lecture 8 - lysosome: mechanisms

Question 1

Draw and explain the sequential steps in creating the M6P tag.

Answer 1

1) GlcNac phosphotransferase binds the lysosomal hydrolase via its signal patch in the cis Golgi.
2) GlcNac phosphotransferase catalyzes the transfer of GlcNac-P (bound to catalytic site) to mannose on the lysosomal hydrolase.
3) In the trans Golgi, a second enzyme cleaves the GlcNac - exposing a M6P signal.
For drawing, refer to notes.

Question 2

Describe and draw the autophagy mechanism.

Answer 2

1) Capture of damaged, obsolete organelles, by crescent shaped membranous structure - the autophagosome.
2) Fusion with lysosome and digestions of the inner membrane and cytosolic contents.

Question 3

Draw and describe the Cholesterol uptake pathway.

Answer 3

1) Cholesterol is transported in the blood as LDL.
2) LDL receptor on cells binds LDL
3) Mono-ubiquitination of receptor cytosolic tail allows interaction with Clathrin adaptor proteins.
4) LDL receptor with LDL is internalized via clathrin coated vesicles
5) Vesicles fuse with early endosome
6) Receptor is recycled back to cell surface (vesicles go back to cell surface)
7) LDL remains in early endosome - transferred to lysosome
8) Eventually, cholesterol gets out of lysosome - transported to ER and gets incorporated into membranes.
For drawing, refer to notes.

Question 4

Describe the steps that occur in a person that has hypercholesterolemia, for cholesterol uptake.

Answer 4

1) Cholesterol transported in the blood as LDL
2) LDL receptors on cells binds to LDL
3) LDL receptor lacks part of cytosolic tail that normally is ubiquitinated
4) Receptors cannot interact with clathrin coat adaptor proteins
5) Receptors cannot be internalized
6) Cholesterol accumulates in blood.

Question 5

Describe the pathway from early endosome to late endosome, and draw it.

Answer 5

1) ubiquitin is covalently attached to the cytosolic tail of the EGF receptor
2) Ubiquitin acts as a signal for the internalization of the receptor
3) Vesicles from early endosomes fuse and migrate along microtubules as multivesicular bodies.
4) The multivesicular body fuses with a late endosome or lysosome.
5) Ubiquitin acts as a signal to promote pinching off and sequestration of entire receptor within lysosome (ESCRT complex).
For drawing the pathway, refer to notes.

Question 6

Describe the ESCRT pathway and draw it.

Answer 6

1) ESCRTs bind endosomal PIPs and ubiquitin.
2) ESCRTs function to cluster receptors and bring them to ESCRT-III, which is one of the ESCRT complexes that mediates invagination.
3) The result is a membrane bound “vesicle” inside the endosome