Lecture 13- using yeast to understand cell membrane trafficking pathways

Question 1

Why do eukaryotic cells need membrane trafficking?

Answer 1

• Compartmentalisation allows more complexity
• Enzymes can modify specific subset of proteins in certain environments
• For sequential modifications, proteins need to be exposed to distinct set of enzymes
• Helps retrieve proteins back to their resident compartment

Question 2

Outline the key steps in the membrane trafficking pathway

Answer 2

1. Protein synthesis on bound ribosomes; cotranslational transport of proteins into/across ER membrane
2. Budding and fusion of ER to Golgi vesicles to from cis-Golgi
3. Retrograde Golgi to ER transport
4. Cisternal progression
5. Retrograde transport from later to earlier Golgi cisternae
6. Regulated and constitutive secretion
7. Sorting to lysosomes
8. Endocytosis

Question 3

How can proteins be modified during membrane trafficking pathways

Answer 3

Can be glycosylated by addition of oligosaccharides and proteolytically cleaved as they transit the ER and golgi

Question 4

How are oligosaccharides added and processed during transit of secretory pathway?

Answer 4

1. In the ER lumen: addition of pre-formed oligosaccharides to an asparagine amino acid in a consensus sequence
2. In the Golgi: the oligosaccharide group is trimmed
3. Later in the Golgi: further sugars are added and the structure can be further branched

Question 5

What issues may occur due to addition of sugars to proteins?

Answer 5

Adds mass which can reduce protein flexibility and affect folding. Reduces proteins ability to be trafficked to membrane

Question 6

What is the purpose of glycosylation?

Answer 6

1. To assist folding
2. Intracellular for trafficking and sorting
3. Outside the cell for interacting with the ECM and other proteins/sugars on other cells

Question 7

List the advantages of yeast as a model organism?

Answer 7

1. Amendable for genetic studies (can be grown as haploid/diploid)
2. Entire genome sequence is known
3. Cheap and easy to grow large quantities
4. Limited gene diversity (both good/bad)
5. Fundamental pathways are highly conserved

Question 8

List the disadvantages of yeast as a model organism

Answer 8

1. Limited cell-cell contact so unlikely to be informative about multicellularity
2. Small so high resolution imaging of intracellular compartments is difficult
3. Cell wall can preclude some types of studies

Question 9

What is the aim of a sec screen?

Answer 9

Aims to identify genes which when mutated cause a defect in the capacity of a cell to secrete proteins to the cell surface

Question 10

What is the aim of end screens?

Answer 10

Aims to identify genes which are involved in endocytosis

Question 11

What is the aim of Vps screens?

Answer 11

Aims to identify genes involved in trafficking proteins which are normally destined for lysosome

Question 12

What was the aim of the sec screen experiment in 1980?

Answer 12

Aimed to investigate secretory pathway in yeast

Question 13

Outline the key points of the sec screen experimental analysis in 1980

Answer 13

1. Cells were analysed for their ability to secrete invertase and acid phosphatase
2. Secretory mutants were defined as strains which failed to export invertase and acid phosphatase
3. But mutants continued to synthesise proteins under restrictive growth conditions
4. By electron microscopy, alternation in the normal ultra-structure of cells could be observed

Question 14

How many genes were identified in the yeast sec screen and what does this tell us about secretion?

Answer 14

23 genes

• At least 23 gene products are required to ensure the transport of proteins from the ER to the PM

Question 15

Why weren’t all the genes/proteins involved in the exocytic pathway identified in the 1980 sec screen?

Answer 15

1. Only identified temperature sensitive mutants
2. Only considered secretion to the PM and not to the endoscome or vacuole
3. Any ‘redundantly’ functioning genes would not have been identified

Question 16

What decision is made in the TGN?

Answer 16

Decision whether to traffic proteins to the PM or the lysosome

Question 17

What is endocytosis?

Answer 17

The process where PM invaginates into the cell, producing a vesicle that is able to fuse with endosomes and enter the endo-lysosomal membrane system

Question 18

What is endocytosis important for?

Answer 18

1. Retrieval of molecules that formed part of the secretory vesicle for recycling
2. Downregulation of signals
3. Remodelling cell surface lipid and protein composition

Question 19

What are the stages in the endocytic pathway?

Answer 19

1. PM to endocytic vesicle
2. Endocytic vesicle to early endosome
3. Early endosome to late endosome to late endosome OR recycling to the PM
4. Late endosome to Golgi OR vacuole

Question 20

What is the major function of the lysosome?

Answer 20

The degradation of extracellular material taken up by endocytosis and certain intracellular components through autophagy

Question 21

What happens to lysosomes enzymes at the TGN?

Answer 21

They are sorted into a pathway destined for lysosomes rather than the PM to ensure they are kept away from the rest of the cell

Question 22

How are vacuole mutants divided?

Answer 22

Divides into classes depending on the stage at which they appear to block the route to the vacuole

Question 23

What are the 4 possible destinations from the late Golgi?

Answer 23

1. PM
2. Early endosome
3. Late endosome/MVB
4. Vacuole

Question 24

How were VPS genes identified?

Answer 24

Through VPS screens and mutant cells

Question 25

Outline the key steps in the CPY pathway

Answer 25

1. CPY is transported through the ER to the Golgi
2. The transport step requires clathrin and Gga1/2
3. In the late Golgi, CPY is specially recognised by a receptor called Vps10
4. CPY dissociated for Vps10 at the late endosome and is transported to vacuoles where is is cleaved to generate the mature form
5. Vps10 is retrieved to the late Golgi through a specific aromatic based signal in its protein sequence