yeast models in membrane trafficking

Question 1

why is membrane trafficking necessary?

Answer 1

• Compartmentalisation allows more complexity for a cell
• Enzymes can modify specific subsets of proteins in certain environments e.g through glycosylation and proteolytic cleavage
• For sequential modifications, proteins need to be exposed to distinct sets of enzymes
• retrieval of proteins back to their ‘resident’ compartment

Question 2

Secretory/Exocytic pathway:

Answer 2

(biosynthetic)
proteins are modified as they travel through
ER → Golgi → PM/endosome/lysosome

Question 3

function of transgolgi network

Answer 3

sorting network to be secreted in right place

Question 4

Endocytic pathway:

Answer 4

(recycling or degradative)
cell surface → endosome → Golgi/ER/lysosome

Question 5

how are proteins modified in er/golgi

Answer 5

oligosaccharide addition (many sugars)
Both N-(asparagine) and O-(OH) linked glycosylation can occur on proteins
asparagines = sites of modification

Question 6

effect of oligosaccharide addition on proteins

Answer 6

sugars have big impact on overall protein structure due to weight - changes conformation

Question 7

function of glycosylation

Answer 7

• assist in folding
• as a ligand = intracellular for trafficking and sorting
  = Outside the cell for interactions with extracellular matrix and with proteins/sugars on other cells

Question 8

what makes organisms useful to identification of faulty genes in trafficking

Answer 8

• Simplicity - trafficking occurs on a cellular scale so a single celled organism is likely to provide information.
• Analysis of specific types of secretion e.g regulated secretion, would need a model system that is able to perform this function.

Question 9

advantages of using yeast

Answer 9

• amenable for genetic studies (can grow as haploid and diploid cells)
• entire genome sequence known since 1996
• limited gene diversity (both ±)
• fundamental pathways conserved

Question 10

disadvantages of using yeast

Answer 10

• limited cell-cell contact so unlikely to be informative about multicellularity
• small (5µm), so high resolution imaging studies of intracellular compartments is difficult.
• Has a cell wall which can preclude some types of studies
  
  • e.g microinjection

Question 11

role of ERGIC

Answer 11

• material is trafficked here after ER
• the compartment then changes rather than individually trafficked then things are const. secreted

Question 12

key experiment in identifying secretory pathway in yeast

Answer 12

novick and schekman
- stages to investigate
- movement in and out
- things docking with plasma membrane
- no transport = cell density increases

Question 13

how many genes were identified as important in trafficking

Answer 13

• 23 were identified by grouping mutants with similar phenotypes
• so at least 23 distinct gene products are required to ensure the transport of proteins from the ER to the plasma membrane.

Question 14

function of class a sec genes

Answer 14

class a = transport to er

Question 15

function of class b sec genes

Answer 15

budding of vesicles to go to golgi

Question 16

function of class c sec genes

Answer 16

fusion of transport vesicles with golgi

Question 17

function of class d sec genes

Answer 17

transport from golgi to secretory vesicles

Question 18

function of class e sec genes

Answer 18

transport from secretory vesicles to cell surface (docking)

Question 19

where is direction of trafficking decided

Answer 19

trans golgi network

Question 20

what is endocytosis

Answer 20

Endocytosis is 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 endosomes and enter the endo-lysosomal membrane system.

Question 21

importance of endocytosis

Answer 21

• Retrieval of molecules that formed part of the secretory vesicle for recycling - labelling to bring back to specific regions
• Downregulation of signals
• Remodelling cell surface lipid and proteincomposition depending on environment
• endocytosis is also a means of entry into cells for many pathogens and toxins

Question 22

stages of endocytic pathway

Answer 22

1. Plasma membrane envaginates to form endocytic vesicle
2. Endocytic vesicle to early endosome
3. Early endosome to late endosome (MVB) or recycling to the plasma membrane
4. Late endosome to Golgi or vacuole

Question 23

major function of the lysosome (vacuole)

Answer 23

degradation of extracellular material taken up by endocytosis as well as certain intracellular components by a process termed autophagy.

Question 24

how does lysosomal protein sorting occur

Answer 24

The lysosome’s resident enzymes are transported to the lysosome through the secretory pathway. At the late Golgi compartment (Trans Golgi Network), they are sorted into a pathway destined for lysosomes rather than the plasma membrane.

Question 25

what is CPY and why is it used

Answer 25

• Carboxypeptidase Y (CPY) is a vacuolar enzyme normally transported to the lysosome having been trafficked through the ER and Golgi.
• As with a-factor CPY is glycosylated and proteolytically cleaved at different stages. This helps us follow its progress

Question 26

Vacuolar mutants are divided into classes depending on what

Answer 26

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

Question 27

what is a multivesicular body

Answer 27

organelle packed full of material and deposits this into a lysosome

Question 28

destinations of transport from golgi network

Answer 28

1. To plasma membrane
2. To early endosome
3. To late endosome/MVB
4. To vacuole

Question 29

CPY signalling mechanism

Answer 29

1. in late golgi gets recognised by receptor protein (vps10)
2. vps10 is sorted into vesicle to traffic to late endosome
3. when bound is recognised by gga1 and gga2
4. cpy dissociates from vps10 at late endosome and transports to vacuoles where it is cleaved to generate mature form
5. Vps10 is retrieved to the late Golgi through a specific aromatic -based signal in its protein sequence (YSSL, FYVF).
   
   • vps10 recognised by specific tail