YEAST

Question 1

Why do eukaryotic cells need membrane trafficking?

Answer 1

Compartmentalisation allows more complexity.
Ezymes can modify specific subsets of proteins in certain environments.
For sequential modifications proteins need to be expose to distinct set of enzymes.
Membrane trafficking is also important in retrieval of proteins back to their resident compartment.

Question 2

What are the major features of the membrane trafficking pathways?

Answer 2

The secretory pathway (biosynthetic) and the endocytic (recycling or degradative) pathway.

Question 3

What are the components of the membrane trafficking pathways?

Answer 3

1. Protein synthesis occur on ribosomes of the the ER.
2. Binding and fusion of ER- Golgi vesicles to form cis-Golgi.
3. Cis Golgi —Medial Golgi –Trans Golgi – Trans Golgi network
4. From the Trans Golgi Network you can have:
   - regulated secretion
   - constitutive secretion
   - transport vesicles to endosomes
5. You can get also retrograde transport in the opposite direction.
6. You can also get endocytosis

Question 4

What are the advantages of yeast as a model organism?

Answer 4

Can be grown as haploid and diploid cells.
Entire genome sequence is known.
Cheap and easy to grow in large quantities.
Limited gene diversity.
Fundamental pathways are conserved.

Question 5

What are the disadvantages of yeast as a model organism?

Answer 5

Limited cell-cell contact is unlikely to be infomative about multicellularity.
They are small and so imaging is difficult.
Has a cell wall which can make some studies difficult or not representative.

Question 6

Yeast have been helpful in identifying genes in the the trafficking pathways. What are the genes classes in the trafficking pathways?

Answer 6

Sec (secretory) genes
Vps (vacuolar protein sorting) genes
End (endocytotic) genes

Question 7

What experiments were done to find the genes involved in the secretory pathway?

Answer 7

Novik/Schekman
Rationale: if proteins cant be secreted ie cells are sec-, they will accumulate.
Analysis: cells analysed for their ability to secrete enzymes (invertase and acid phosphatase).
The mutants have a different ultra structure that can be seen under a microscope.

Question 8

How many sec mutant genes were found?

Answer 8

23 genes identified by phenotype. This means there are at least 23 distinct gene products that ensure transport of proteins from the ER to the plasma membrane. These were placed into different classes.

Question 9

What are the classes of sec- mutants?

Answer 9

A - accumulation in cytosol
B- accumulation in RER
C- accumulation in ER to golgi vesicles
D- accumulation in Golgi
E- accumulation in secretory vesicles

Question 10

Certain proteins show different modifications through the secretory pathway such as Alpha Factor. How could this be helpful?

Answer 10

Alpha Factor is glycosylated and proteolytically cleaved at different stages through the secretory pathway. This means we can find where in the pathway certain mutant are affecting.

Question 11

Why werent all the genes/proteins in the exocytic pathway identified by Novik and Schekman?

Answer 11

1. They only identified temperature sensitive mutants. Not all genes when mutated will cause this phenotype.
2. They only considered secretion to the plasma membrane so defects in transport to endosome or vacuole would not be identified.
3. Any redundantly functioning genes would not be identified (although yeast has a low redundancy.)

Question 12

What is endocytosis?

Answer 12

The process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with the endosomes and enter the endolysosomal membrane system

Question 13

Why is endocytosis important?

Answer 13

Retrieval of molecules that formed part of the secretory vesicle for recycling.
Downregulation of signalling.
Remodelling of the cell surface composition.

Question 14

What are the stages of the endocytic pathway?

Answer 14

Plasma membrane to endocytic vesicle.
Endocytic vesicle to early endosome.
Early endosome to late endosom (MVB) or recycling to plasma membrane.
Late endosome to Golgi or Vacuole.

Question 15

When screening for end- mutants, what can mutants not do?

Answer 15

Screening uses a fluid marker called lucifer yellow or a bound pheromone alpha factor. End mutants cannot internalise the marker. So the wil type will light up.

Question 16

How many End- mutant were detected through lucifer yellow screening?

Answer 16

7 End- genes, of these, 5 directly involved in the process of membrane invagination and scission.

Question 17

What is the major function of the lysosome(vacuole)?

Answer 17

Degradation of EC material take up by endocytosis. Some IC components are also taken up. These organelles contain degradative enzymes that must be kept separate frmo the rest of the cell.

Question 18

Where do the enzymes of the lysosome come from?

Answer 18

The resident enzyme of the lysosome are transported to the lysosome through the secretory pathway. At the late Golgi they are sorted into a pathway destined for the lysosomes rather than the plasma membrane.

Question 19

What happens in the Vacuolar Protein Sorting mutant screens?

Answer 19

Carboxypeptidase Y (CPY) is normally transported to the lysosome having been trafficked through the ER and Golgi. Like Alpha Factor in the Sec pathway, CPY is glycosylated and proteolytically cleaved at different parts of the pathway. 
Generation of mutagenised cells and observation of the cells that screte CPY.

Question 20

How many genes were identified in the VPS pathways?

Answer 20

Over 60 genes were identified in this way. Many of the genes work in complexes.

Question 21

How are vacuolar mutants divided?

Answer 21

Depending on the stage of blockage.

Late Endosome to MVB is the most common mutation.

Question 22

What determinants tell us where the protein needs to go?

Answer 22

There are four possibilities frmo the late Golgi:

• To the plasma membrane
• to the early endosome
• to the late endosome/MVB
• to the vacole

These are determined by molecular signals.
No signal = to the plasma membrane

Question 23

What is the CPY pathway?

Answer 23

The pathway from Late Golgi to MVB
CPY recognised by Vps10
Vps10 puts transport factors in motion such as clathrin and adpators.
Vps10 is retrieved to the late Golgi though protein signal sequences.