Order of events in the yeast secretory pathway

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Order of events in the yeast secretory pathway

• Novick, Ferro, and Schekman

• What is the question?
• What is their approach?
• 3. What do I need to review to understand this
  paper?

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Whats the question?

• What is the order of events in secretion?

Whats the approach?

• Genetics (with confirmation by microscopy)

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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 22 sec defect
• (what does this indicate? Remember, they had no
  marker on their mutant gene they didnt 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.

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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 arent marked and in
  organisms whose life cycle is almost exclusively
  diploid.

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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.

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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

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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

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Results

• Question Is it possible to detect the
  localization of invertase biochemically, i.e. by
  its processing during secretion?
• Figure 3 shows later mutants have
  increasingly glycosylated invertase.

glycosylated
unmodified
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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.

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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.

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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.

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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.

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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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