L2 - Investigation of Autophagy Flashcards
What was the model system used to investigate the molecular mechanisms of autophagy?
budding yeast
Why is yeast such an effective model for discovering the molecular mechanisms of autophagy?
1) easy and fast to grow/culture
2) small genome for a euk
3) can be diploid or haploid
4) 50% of yeast genes have human orthologues
5) powerful methods for editing genome
In a normal cell, what event triggers increased autophagy?
nutrient starvation
Do yeast cells have a lysosome?
no, but they have a large vacuole that is analogous to lysosome in human cells
How did Ohsumi try and prove that yeast was an effective model for studying autophagy?
Did a genetic screen to find yeast strains deficient in vacuolar proteases
- grew in rich media
- switched to N-starvation media
- examined each mutant by light microscopy
- mutants won’t grow in nitrogen-deficient medium
what is another way to induce autophagy in the vacuoles of wild type yeast cells?
treat cells with protease inhibitor (PMSF) to inhibit activity of proteases inside vacuole
how can you tell that a cell is not able to undergo proper autophagy?
accumulation of autophagic bodies in the vacuole (material is taken up but the vacuole can’t actually digest it)
describe the general process for cloning by complementation
1) break genome into fragments with RE
2) ligate fragments into plasmids to generate library
3) transform into yeast cells
4) screen transformants that can grow in SD-N
5) isolate the plasmid and sequence the DNA
Why was Atg1 so special?
first gene that was found to be related to autophagy
How did they get the first hint on the SPECIFIC function of Apg1?
Had a genome match with the N-term of a protein kinase found in worms/C. elegans
how can autophagic bodies be observed?
1) light microscopy
2) electron microscopy
what is the function of protein kinases?
protein that covalently attached P from ATP to a protein substrate
why were they able to do a secondary genetic screen after finding Atg1?
can now use loss of viability upon starvation as a phenotype instead of AB accumulation as a phenotype?
- allows for much faster screening and can compare directly between SD-N and regular medium
what are the two classes of protein kinases?
1) phosphorylate at Tyr
2) phosphorylation at Ser or Thr
what kind of protein kinase is Unc-51?
phosphorylates Ser and Thr
describe general process of IP
1) lyse cells
2) add Ab that recognizes specific protein in the lysate
3) add conjugated bead that has high specificity for Ab
4) wash away the unbound lysate material and spin down beads
5) do WB analysis
what are the uses for immunoprecipitation?
1) isolate a larger amount of a particular protein from many other proteins
2) identify if there is a protein of interest/quantify amount in a cell lysate
what is Western Blot for?
method to detect specific proteins in a sample
what are the general steps of WB?
1) run samples of SDS-page to separate proteins
2) transfer proteins on gel to membrane
3) add blocking buffer to block any spots not bound to protein
4) add primary Ab solution
5) wash unbound Ab
6) add secondary Ab with HRP
7) develop blot (add chemiluminescent reagent that can be cleaved by HRP on secondary Ab to produce luminescence)
8) place film against membrane to detect light from the reaction
what are the membranes that are used in WB?
1) nitrocellulose
2) PVDF
HRP
horseradish peroxidase
- a moiety commonly found on secondary Abs used in WB to produce luminescence for detection of a primary Ab
what was the method used to prove that that Atg 1 was a protein kinase?
In vitro kinase assay
describe the process used to prove the kinase activity of Atg1?
1) WB with anti-myc Ab with myc-tagged Atg1 to detect presence of Atg1 isolated
2) SDS-page
3) transfer proteins to membrane to isolate Atg1
4) autoradiography with kinase, substrate and Y32P-ATP to detect the amount of radioactivity which will correlate to the amount of phosphorylation occurring
why did the sequence of the other Atg proteins not really help in figuring out there functions?
the sequences of these other Atg proteins did not have any sequence similarities with other known proteins at the time
