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Order of events in the yeast secretory pathway

Order of events in the yeast secretory pathway. What is the question? What is their approach? 3. What do I need to review to understand this paper?. Novick, Ferro, and Schekman. What’s the question?. What is the order of events in secretion?. What’s the approach?.

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Order of events in the yeast secretory pathway

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  1. Order of events in the yeast secretory pathway • What is the question? • What is their approach? • 3. What do I need to review to understand this paper? Novick, Ferro, and Schekman

  2. What’s the question? • What is the order of events in secretion? What’s the approach? • Genetics (with confirmation by microscopy)

  3. What is a classical genetics approach for ordering genes/proteins that appear to be in the same pathway? • Have complementation groups with similar phenotypes, i.e. no invertase secretion, but with three different morphologically identifiable defects. • Mate haploids, sporulate, identify tetrads in which there is 2:2 sec defect • (what does this indicate? Remember, they had no marker on their mutant gene – they didn’t even know where they were) • Epistasis • Gene A acts upstream of gene B and has phenotype A (gene B has phenotype B). A double mutant will have phenotype A because gene A is epistatic to gene B.

  4. Is this still a valid approach and why? • It is one of the ways we test the order of genes even in sequenced organisms. • It is harder if the genes aren’t marked and in organisms whose life cycle is almost exclusively diploid.

  5. Ordering conditional mutants • If two mutations have different non-permissive conditions, it is possible to order them by shifting from one to the other and looking for maintenance of the mutant phenotype. • In this case, all the mutations were TS, so a non-genetic test, e.g. microscopy, was the only way. • Leaky mutations would cause problems here.

  6. Assay/phenotype • Could detect invertase in 10 minutes after induction and specific activity increased at a constant rate for 30 minutes. • If they added cycloheximide, secretion continued for 5 minutes, therefore, secretion takes about 5 minutes

  7. Results • sec18 – ER and some 40-60 nm vesicles • sec7-1- Berkeley bodies • sec1-1 – 80-100 nm vesicles • Conclusion: • All of these, when mated to sec18 mutants, showed the sec18 phenotype. • Most, when mated to sec7, produced Berkeley bodies (bB) • Order: ER and small vesicles/Bb/larger vesicles

  8. Results • Question: Is it possible to detect the localization of invertase biochemically, i.e. by it’s processing during secretion? • Figure 3 – shows later mutants have increasingly glycosylated invertase. glycosylated unmodified

  9. Results • Question: Can these mutants be used to test drugs for their effects on secretion independent of their effect on protein synthesis? • Tried DNP (uncoupler), reduces available ATP. Tried other drugs – A23187, a calcium ionophore – everything that they tried that blocked secretion affected ATP and also worked in wt cells. • Yes – since these mutants secrete invertase in the presence of protein-synthesis inhibitors.

  10. Results • Question: Why did some mutants secrete more at 250C with glucose and DNP than with glucose alone (Table 2)? • (Figure 4) – at high levels of glucose, in the absence of DNP, secretion was inhibited. This was seen only with sec7 mutants. • With very low glucose, sec7 mutants accumulate Golgi-like structures. With high glucose – Berkeley bodies. Conclusion: there is a Golgi block that is affected by glucose.

  11. Results • Question: Do energy-requiring and sec protein functions occur sequentially or concurrently? • How would you test this? • Epistasis-like experiment – block then temperature shift, temperature shift, then block. Do vesicle profiles change? • No. Therefore, energy and sec functions are concurrent.

  12. Discussion and conclusions • Established in a general sense, the sequence of events. • Sec mutants that are not very thermoreversible function late in the pathway, i.e. accumulate 100 nm vesicles (sec3,4, and 5) • Looked at glycosylation – found oligosaccharides only on Golgi-blocked invertase. This was the beginning of our understanding of the post-translational processing of secreted proteins. • ATP vs membrane potential, other effect of DNP-like drugs – now they had a system to test this.

  13. Discussion/conclusions • Energy required for at least three steps in secretion (ER and 40-60 nm vesicles, Golgi, and 100 nm vesicles. • Glucose has a specific effect on sec 7. Then Now

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