YEAST - the endocytic pathway

Question 1

Why is sorting to the endosome needed?

Answer 1

• For degradation of material taken up by endocytosis

- Lysosomes contain enzymes that break down this material

Question 2

Carboxypeptidase Y protein - how is it used in Yeast vacuolar sorting screens?

Answer 2

Carboxypeptidase Y is an enzyme which cuts peptide bonds at the carboxy terminus. It is normally transported from the lysosome to the Golgi.

This tends to not happen in mutant cells.

Question 3

How is vacuolar sorting screening analysis carried out?

Answer 3

Microscopy – flourescence and electron

Biochemical - western blotting. This identified that a propeptide is cleaved away at the vacuole

Question 4

How are lysosomal sorting mutants classified?

Answer 4

Genes mutated in these cases have been IDENTIFIED and GROUPED based on the secretion stage that is BLOCKED in the mutation.

Grouped A-F depending on appearance

Question 5

What are the possible trafficking destinations of cargo from the late Golgi?

Answer 5

• Plasma membrane
• Lysosome/Vacuole (yeast)
• Early endosome
• Late endosome

Question 6

What is ENDOCYTOSIS?

Answer 6

‘Process by which the plasma membrane invaginates inwards and a vesicle is produced - these vesicles can fuse with endosomes and enter the lysosomal membrane system’

Question 7

Why do we need endocytosis?

Answer 7

• Reduction in protein signalling
• Regulation of cell surface composition
• Retrieval of signalling molecules
• ALSO MEANS OF ENTRY FOR VIRUSES AND TOXINS

Question 8

How have studies demonstrated endocytosis?

Answer 8

Differences in density between the early and late endosome
Microscopy of mammalian cells
Yeast genetic studies

Question 9

Outline the CpY pathway (carboxypeptidase Y) in YEAST

Answer 9

Cpy produced and transported in its pre-pro form from the ER–> Golgi
Recognised by a receptor in the late Golgi
Receptor binds to CpY and the complex is recognised by cytoplasmic factors Clathrin and Gga1/2
CpY dissociates from the receptor at the late endosome
It is transported to the vacuole where it is cleaved to an even shorter form
The receptor is retrieved back to the late Golgi via a SPECIFIC AROMATIC SIGNAL

Question 10

Outline the ALP (alkaline phosphatase) pathway

Answer 10

ALP protein controls sorting from the Golgi to Vacuole
Has acidic sorting signal domains 
Requires AP3 (adaptor protein)

Question 11

What is KEX2 and why is it needed? What happens to it to allow its function?

Answer 11

Mating pheromone processing enzyme

Can be phosphorylated — needed for transport from the early to late Golgi

Question 12

STEPS IN ENDOCYTOSIS

Answer 12

Plasma membrane – endocytic vesicle – early endosome – late endosome – either PLASMA MEMBRANE or VACUOLE/LYSOSOME

Question 13

How can this be visualised in YEAST?

Answer 13

VISUAL LOCALISATION

END SCREEN - add liquid phase marker (luciferase) and see how it is internalised by cells

Question 14

How do the early and late endosomes differ from each other?

Answer 14

Compartments have different densities

Question 15

Features of the late endosome

Answer 15

Receives material from both the EXO and ENDOCYTIC pathways

Sorting to here involves SIGNALS which sorts components on the surface of the late endosome into MVBs

Question 16

What are ESCRTS?

Answer 16

Sorting signals that sort componants on the surface of the late endosome into MVBs

Question 17

What are MVBs and how are they formed?

Answer 17

Multi-vesicular bodies – they form when endosomes THEMSELVES invaginate inwards forming internal vesicles,
The MVB fuses with the vacuole to deliver vesicles for degradation