Lecture 2 Flashcards
4 problems with autophagy?
- Experience with lysosomes
- Autophagosomes are transient (appears/dissapears)
- Em methods - not quantitative
- No “marker” for autophagosomes to label autophagosomes,
5 reasons why budding yeast is used to investigate the molecular interactions of cellular pathways?
- Microbe: Cheap and easy to grow
- Diploid and Haploid states
- Eukaryote with very small genome, genome is fully sequenced
- 50% yeast genes- orthologous; early predecessor and similarity in pathways.
- Powerful genetics (receptive to changing genome dynamically): investigate biological processes, yeast as turning point in autophagy pathway.
How did Ohsumi use a yeast genetic screen to discover genes involved in autophagy?
- Vacuole. Usually multiple copies of lysosome within cytoplasm. Phenotype: accumulation of autophagic bodies (vacuole) upon Nitrogen starvation (normal).
Process of Ohsumi’s genetic screen?
Generate a mutant yeast strain, “delete” or remove a vacuolar proteinase gene. Grow in rich media (YPD) (WT and mutant). Transfer/switch media (Nitrogen starvation media; SD-N). Examine each mutant through light microscopy, look for loss of normal phenotype (loss of autophagic body accumulation).
Steps in cloning by complementation?
Yeast genomic DNA (WT) — restriction endonuclease — genomic DNA fragments — ligation into plasmids — genomic library — transformation into apg1-1 mutant strain – transformed yeast cells — screen for the transformations that can grow in nitrogen depleted media (SD-N) — Isolate plasmid for DNA sequencing.
How does protein level change in the cell?
- Transcription regulation
- Protein synthesis (translation control +protein folding)
- Protein turnover (breaking down proteins into individual amino acids)
- Folding
What network is used o ensure proteostasis?
- Translation initial factors and ribosomes ensures efficient translation (generation of polypeptide)
- Molecular chaperones enables a protein to adopt its native structure allowing it to perform its physiological function.
- Recycling/breakdown systems - turning over misfolded, aggregated, aged proteins.
Why study protein recycling?
- Protein folding is complex, not 100% effective
- Proteins in cells have half-lives
- Metabolic control
What is the problem with UPS and chaperone network?
Larger objects
How is autophagy affected by glucagon and insulin?
Glucagon induces autophagy, insulin inhibits autophagy
What are the technical challenges in studying autophagy?
- Lysosomes are heterogenous in shape and content (requires experience).
- No good method to quantitate extent of autophagy.
- Autophagosome is a transient organelle (exists for 10 minutes before fusion with lysosome).
- Lack of specific “marker” to track autophagosome for biochemical studies.
What were the results of the 1st genetic screen of autophagy?
- Identification of mutant allele apg1
- Apg1 mutant lost viability faster than WT cells in nitrogen-deficient medium.
- Loss of viability promotes secondary genetic screen.
Benefits of secondary genetic screen?
- Screens mutants much more quickly.
- With the replica plating method, one is able to compare the ability of different mutants to growth on nitrogen starvation medium and regular medium through replica plating method.
- Isolated apg2-apg15
Procedure: Cloning of 1st autophagy gene Apg1 (Atg1)
- Isolate genomic DNA from WT yeast cells
- Prepare genomic library by restriction digestion and cloning fragments into vector.
- Transform library of plasmids into apg1-1 mutant strain
- Look for transformants that enable ap1-1 mutant to grow in nitrogen-deficient media (ex. reversing phenotype)
- Isolate plasmid, sequence fragment of genome.
Function of protein kinase
Enzyme covalent attaches phosphate group from ATP to protein substrate.
