polarised secretion in yeast Flashcards
How were components of the sec pathway determined?
mutant screens
What mutants were they screening for when looking for secretory pathway defective mutants?
Cells with increased cell density - lipid and protein synthesis still occurs but cell division stop
How were dense cells separated in the mutant screens?
Using cell density gradient centrifugation after random mutagenesis
What type of mutation is secretory mutation?
lethal mutant (so mutant screens had to be ts)
What two enzymes did they screen for the release of to assay secretion pathways?
invertase and acid phosphatase
What gene found in post Golgi vesicles?
Sec4 - a RAB GTPase
Where can sec4 be found?
Localised to bud tips
If you create a sec4 mutant with other late sec temp sensitive mutants, what is the result?
Synthetically lethal mutant (eg even in permissive temp will dieee)
What three components is the localisation of sec4 dependent on?
Sec2, Actin and Myosin
How does Sec2 act?
Sec 2 is a GEF to Sec4, required to localise Sec4 on the post Golgi vesicles
What proteins are required for actin cable assembly?
Formins
Which two formins act in polarisation of secretion pathway
Bri1/Bnr (redundantly)
How can you engineer mutants to study formin proteins?
knockout either bri1 or Bnr and then make the other a temperature sensitive mutation
How can you disrupt actin cable assembly?
Knockout TMP1 gene and temperature sensitive TMP2 gene
What are TMP encoding?
tropomyosin
