yeast screens

Question 1

Why do eukaryotic cells need membrane trafficking?

Answer 1

¥ Compartmentalization allows more complexity.
¥ Enzymes can modify specific subsets of proteins in certain environments e.g glycosylation and proteolytic cleavage.
¥ For sequential modifications, proteins need to be exposed to distinct sets of enzymes.
Membrane trafficking is also important in retrieval of proteins back to their ‘resident’ compartment

Question 2

The major features of membrane trafficking pathways

Answer 2

Secretory/Exocytic (biosynthetic) pathway: ER to Golgi to PM/endosome/lysosome

Endocytic pathway (recycling or degradative): cell surface to endosome to Golgi/ER/lysosome

Question 3

Yeast as a model organism

Advantages:

Answer 3

amenable for genetic studies (can grow as haploid and diploid cells)

entire genome sequence known since 1996 (and is fully annotated),cheap and easy to grow in large quantities (good for biochemical studies),

limited gene diversity (both ±)

fundamental pathways conserved

Question 4

Yeast as a model organism

disadvantages

Answer 4

Disadvantages:

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

Question 5

Novick and Schekman 1980 experiments

what was the aim of their experiments

Answer 5

Aim: to investigate the secretory pathway in yeast.

Question 6

Novick and Schekman 1980

what enzymes were studied in the Sec- analysis done

Answer 6

cells analysed for their ability to secrete enzymes invertase and acid phosphatase

Question 7

what are secretory mutants

Answer 7

secretory mutants as those strains which fail to export active invertase and acid phosphatase, but continued to synthesize protein under restrictive growth conditions

Question 8

what was seen in sec mutants

Answer 8

1 alterations in the normal ultra-structure of cells could be observed e.g accumulation of vesicles or aberrant membranous structures. Secretory mutant cells have a different ultra-structure

Question 9

How many sec genes were identified

Answer 9

23 distinct gene products are required to ensure the transport of proteins from the ER to the plasma membran

Question 10

how did they split the Sec- genes into the 5 classes?

Answer 10

Mutant groups were placed in sequential order by combining mutants from different classes and by use of more detailed analysis of protein modifications

Question 11

Class A - fate of secreted proteins

Answer 11

accumulate in cytosol as cannot be transported into the ER

Question 12

Class B - fate of secreted proteins

Answer 12

accumulate in rough ER as budding of vesicles from ER doesn’t occur

Question 13

Class C - fate of secreted proteins

Answer 13

accumulation i ER-to-Golgi transport vesicles as no fusion of vesicles to golgi

Question 14

Class D - fate of secreted proteins

Answer 14

accumulation in golgi as no transport from golgi to secretory vesicles

Question 15

Class E - fate of secreted proteins

Answer 15

Accumulation in secretory vesicles as transport to cell surface does not occur

Question 16

Why weren’t all of the genes/proteins involved in the exocytic pathway identified by Novick and Schekman

Answer 16

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 though yeast has relatively low gene redundancy which underpinned the success of these approaches.

Question 17

what do Sec screens show

Answer 17

mutations affecting ER-plasma membrane but proteins are also synthesized and need to be trafficked to the lysosome. A decision is made in TGN whether to traffic to surface or towards lysosome.

Question 18

What is Endocytosis ?

Answer 18

process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle

Question 19

Why is endocytosis important

Answer 19

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

Question 20

Stages in the endocytic pathway

Answer 20

Plasma membrane to endocytic vesicle

Endocytic vesicle to early endosome

Early endosome to late endosome (MVB) or recycling to the plasma membrane

Late endosome to Golgi or vacuole

Question 21

End- screens looked for … and what did it show the importance of

Answer 21

mutants that could not internalise a fluid phase marker (lucifer yellow) or a bound pheromone alpha-factor.

identification indicated importance of actin cytoskeleton in yeast endocytosis

Question 22

major function of the lysosome (vacuole

Answer 22

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

Question 23

Carboxypeptidase Y (CPY) normal transport route

Answer 23

ER - golgi - lysosome

Question 24

Carboxypeptidase Y (CPY) how is it modified at different stages and what receptors or cytoplasm factors are required

Answer 24

• CPY is glycosylated (has long sugar chains added) and proteolytically cleaved at different stages this helps us follow its progress
• CPY is synthesized in a prepro form and is transported through the ER to the Golgi.
• Sorting: in the late Golgi CPY is specifically recognised by a receptor Vps10 i.e sorting is receptor-mediated.
• The transport step requires cytoplasmic factors: clathrin and two adaptors called Gga1 and Gga2.
• CPY dissociates from Vps10 at the late endosome/MVB and is transported to vacuoles where it is cleaved to generate the mature form.
• Vps10 is retrieved to the late Golgi through a specific aromatic -based signal in its protein sequence (YSSL, FYVF).

Question 25

4 destinations possible for trafficking from the late golgi

Answer 25

Four possible destinations

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