lec 13. yeast screens

Question 1

Exocytic/Secretory (biosynthetic) pathway

Answer 1

ER -> Golgi -> endsome/lysosome/PM

Question 2

Endocytic (recycling/degradative) pathway

Answer 2

cell surface -> early endosome -> late endosome (MVB) -> Golgi/ER/Vacuole(lysosome)

OR

cell surface -> early endosome -> recycling to PM

Question 3

are mitochondria part of secretory pathway

Answer 3

no!

Question 4

organisms used in identifying trafficking genes

Answer 4

mouse, drosophila, c. elegans, zebrafish, yeast

Question 5

advantages of yeast as model organism

Answer 5

can grow as haploid and diploid, know entire genome, cheap, have conserved pathway

Question 6

disadvantages of yeast as model organism

Answer 6

has a cell wall, small size makes imaging difficult, not good for multicellularity

Question 7

Sec Screen

Answer 7

what might happen if secretion is disrupted?
mutants will be more dense as vesicles accumulate, and cell continues to synthesize proteins

23 genes needed for transport from ER -> PM

Question 8

how did they order the actions of genes in trafficking pathways

Answer 8

they combined mutants to identify where a mutation acts

EX. in sec screen, by studying size of alpha factor in different mutants

Question 9

Alpha factor

Answer 9

pheromone secreted by budding yeast

• glycosylated in ER
• extra glycosylated in Golgi
• proteolytically cleaved at different stages

Question 10

classes of Sec genes by defective function

Answer 10

A - transport to ER
B - budding of vesicles from RER
C - fusion of vesicles with Golgi
D - transport from Golgi to secretory vesicles
E - transport from secretory vesicles to cell surface

Question 11

why did they not identify all the Sec genes?

Answer 11

1) only identified temperature sensitive mutants
2) only looked at transport to PM, not endosome/vacuole
3) did not identify redundantly functioning genes as yeast has few of these

Question 12

endocytosis

Answer 12

plasma membrane invaginates, producing a vesicle thats able to fuse with endosome and enter the endo-lysosomal system

Question 13

End Screen

Answer 13

what would happen if endocytosis is disrupted?
mutants would be unable to internalise fluid phase marker (lucifer yellow) or bound alpha factor

7 genes needed for membrane invagination + scission

Question 14

lysosomes

Answer 14

contain certain degradative/proteolytic enzymes for degradation of extracellular material (from endocytosis) or intracellular material (autophagy). Its resident enzymes are sorted in the late golgi compartment into pathway destined for lysosome, not PM

Question 15

Vps Screen

Answer 15

what would happen if material didn’t get sorted into vacuoles/lysosome?
Mutants secreted CPY, which normally goes into lysosome

60 vacuolar protein sorting genes identified

Question 16

Carboxypeptidase Y (CPY)

Answer 16

• PI formed in ER
• lightly glycosylated in Golgi
• proteolytically cleaved in vacuole

recognised by Vps10 in late golgi then transported into late endosome (transport requires clathrin, Gga1, Gga2). Vps10 recycled backinto late gogli, while CPY is transported into vacuole

Question 17

4 possible destinations for proteins in TGN

Answer 17

1) plasma membrane
2) early endosome
3) late endosome
4) vacuole