Lecture 34: Function and regulation of autophagy

what is autophagy?

an intracellular self-degradative process found in all eukaryotic cells

what is the role of autophagy in normal cell function?

Clearance of cytoplasm (basal):

• removal of aged ribosomes, abnormal protein aggregates, long-lived proteins, invasive viruses, etc.

• turnover of excessive/defective/harmful organelles

what is the role of autophagy in stress response?

Nutrient recycling (induced):

in response to new cell development need, starvation, and other stresses it provides

• amino acids for protein synthesis

• energy source for cell development or survival

what is a cell a balanced state between?

synthesis and degredation (replacement of proteins every 3 months)

new central dogma

DNA → RNA → Protein → folding, trafficking, turnover

general process of autophagy

omegasome (induction & nucleation) → phagophore → autophagosome (expansion/maturation) → autolysosome (fusion of autophagosome with lysosome) → degradation and recycling

Ohsumi’s genetic screen for yeast apg (atg) mutants

1. treat cells with a point mutation (EMS), ~38000

2. first screen for loss of viability in N starvation (wild type survive, ~ 2700 mutants don’t)

3. second screen for lack of autophagic body accumulation in vacuole/ (99 mutants)

4. complementation analysis (adding wild-type gene back to mutant to see if it changes)

5. 14 remaining mutants = 14 Apg mutants isolated

what is Atg1?

a kinase that induces autophagy

what happens to Atg1 in conditions where autophagy is favoured?

the protein complex with Atg13 dephosphorylates and fuses to Atg1, and is targeted to the omegasome

what allows phagophores to grow?

lipidated Atg8/ LC3-II

what is used to attach lipids to Atg8?

Atg12-Atg5-Atg16 complex

important note about Atg8/LC3

when on growing phagophores they serve as a cargo ligand that recruits cargoes (ex: defective organelles)

what is autophagy in the mitochondria called?

mitophagy

who supplies membranes for phagophore expansion?

Atg9 vesicles (from ER) and Atg2/Atg18 channels (direct contact with ER)

how is Atg18 tethered onto the ER?

It is attached to Rab18, acting as a tethering factor, and when Rab18 goes through GTP hydrolysis it releases the phagophore

what regulates fusion of autophagosomes with lysosomes?

Atg8 / LC3 needs to be released from the autophagosome to allow for fusion:

• SNAREs: SNAP29 (and other?)

• Rab proteins: Rab7 (?)

• Tethering factors: Atg 14

when specifically is autophagy transiently induced and why

immediately following birth to enable newborn to obtain nutrients before it is able to drink from mom and after removal of placenta

what happens to newborns if you knockout Atg5?

there is no lipidation of Apg8, meaning that there is no induced autophagy, leading to no nutrients

how is autophagy used in viral infections?

to clean out pathogens

explain process of Sars-Cov-2 in autophagy

1. spike proteins are recognized by receptors on cell membrane

2. endocytosis to early endosome

3. membrane fusion to late endosome, releasing virus gene

4. virus gene within autophagophore, should go to the lysosome but the protein released (ORF7a) blocks fusion

5. formation of DMV (double membrane vesicle)

6. Release of new virus

what inihbits ORF7a?

Glecaprevir, allowing for normal pathway to continue