L2 - Investigation of Autophagy

Question 1

What was the model system used to investigate the molecular mechanisms of autophagy?

Answer 1

budding yeast

Question 2

Why is yeast such an effective model for discovering the molecular mechanisms of autophagy?

Answer 2

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

Question 3

In a normal cell, what event triggers increased autophagy?

Answer 3

nutrient starvation

Question 4

Do yeast cells have a lysosome?

Answer 4

no, but they have a large vacuole that is analogous to lysosome in human cells

Question 5

How did Ohsumi try and prove that yeast was an effective model for studying autophagy?

Answer 5

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

Question 6

what is another way to induce autophagy in the vacuoles of wild type yeast cells?

Answer 6

treat cells with protease inhibitor (PMSF) to inhibit activity of proteases inside vacuole

Question 7

how can you tell that a cell is not able to undergo proper autophagy?

Answer 7

accumulation of autophagic bodies in the vacuole (material is taken up but the vacuole can’t actually digest it)

Question 8

describe the general process for cloning by complementation

Answer 8

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

Question 9

Why was Atg1 so special?

Answer 9

first gene that was found to be related to autophagy

Question 10

How did they get the first hint on the SPECIFIC function of Apg1?

Answer 10

Had a genome match with the N-term of a protein kinase found in worms/C. elegans

Question 11

how can autophagic bodies be observed?

Answer 11

1) light microscopy

2) electron microscopy

Question 12

what is the function of protein kinases?

Answer 12

protein that covalently attached P from ATP to a protein substrate

Question 13

why were they able to do a secondary genetic screen after finding Atg1?

Answer 13

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

Question 14

what are the two classes of protein kinases?

Answer 14

1) phosphorylate at Tyr

2) phosphorylation at Ser or Thr

Question 15

what kind of protein kinase is Unc-51?

Answer 15

phosphorylates Ser and Thr

Question 16

describe general process of IP

Answer 16

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

Question 17

what are the uses for immunoprecipitation?

Answer 17

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

Question 18

what is Western Blot for?

Answer 18

method to detect specific proteins in a sample

Question 19

what are the general steps of WB?

Answer 19

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

Question 20

what are the membranes that are used in WB?

Answer 20

1) nitrocellulose

2) PVDF

Question 21

HRP

Answer 21

horseradish peroxidase

- a moiety commonly found on secondary Abs used in WB to produce luminescence for detection of a primary Ab

Question 22

what was the method used to prove that that Atg 1 was a protein kinase?

Answer 22

In vitro kinase assay

Question 23

describe the process used to prove the kinase activity of Atg1?

Answer 23

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

Question 24

why did the sequence of the other Atg proteins not really help in figuring out there functions?

Answer 24

the sequences of these other Atg proteins did not have any sequence similarities with other known proteins at the time

Question 25

what was the rate-limiting step of autophagy?

Answer 25

autophagosome formation

Question 26

PAS

Answer 26

preautophagosome structure, origin of and autophagosome in yeast cells fond next to vacuole

Question 27

describe fluorescence-based protein localization

Answer 27

1) genetically fuse a fluorescent protein to Atg genes and track their location via fluor microscopy

Question 28

what are the common fluor proteins used?

Answer 28

1) GFP
2) CFP
3) YFP

Question 29

what were the important findings of the fluor microscopy experiment for early Atg research?

Answer 29

Found 18 Atg proteins localize to the PAS upon starvation when autophagy is induced

Question 30

why did we care about these 18 Atg proteins that were found in yeast?

Answer 30

most conserved across other species and thought to be a part of a core autophagy machinery

Question 31

what was the findings of the second fluor microscopy experiment done in early autophagy research?

Answer 31

found that the Atg proteins function in a hierarchical fashion (some depended on the other)

Question 32

what are the 5 Atg functional groups?

Answer 32

1) Atg1 and regulators
2) Ptdlns 3-kinase complex
3) Atg12 conj sys
4) Atg8 conj sys
5) Atg2-Atg18 complex and Atg9

Question 33

what was the quantitative autophagy assay that was developed?

Answer 33

ALP assay using the alkaline phosphatase Pho8 protein

• normally Pho8 gets delivered to ER
• needs its N-term to get to ER for vacuole transport, so Pho8Δ60 stays in cytosol and only transported to vacuole via non-specific autophagy
• inactive Pho8Δ60 gets engulfed with cytoplasmic material during autophagy
• Pho8Δ60 protein active in vacuole and dephosphorylates a substrate that becomes fluorescent

Question 34

proteinase B

Answer 34

a protein that activates Pho8 by cleavage of C-term of Pho8 (assuming that it is found in the vacuoles)

Question 35

how is Pho8 activated in the vacuole?

Answer 35

by a proteolytic enzyme proteinase B in vacuole

Question 36

prb1

Answer 36

gene that encodes for proteinase B, the protein that cleaves Pho8 for its activation

Question 37

can ALP assay be done with living yeast cells?

Answer 37

Yes!?