2.  Secretory pathway- the process of moving proteins out of the cell through a series
a steps (secretion).
 The path of the protein origins are in the rough endoplasmic reticulum.

3.  Invertase and acid phosphatase fail to be secreted in a new class of mutants that
are temperature-sensitive for growth (class a secretory mutants).
 class b mutants do not produce active secretory enzymes at the restrictive
temperature (37 degrees celsius).

4.  Density enrichment procedure
 Invertase immunoprecipitation
 Glycosylated forms of invertase
 Cpy immunoprecipitation
 Enzyme assays

5.  Only 190 accumulated active invertase at 37 degrees celsius.
 34 complementation groups
 Screened for defects in protein synthesis
 Only 5 of the groups synthesized protein at 50% or more of the wild type (x2180-1a) rate at 37
degrees celsius.
 The constitutive form of invertase is not a precursor of the secreted enzyme.
 All 5 of the secretory mutant candidates secreted nearly normal levels of invertase at 24 degrees
celsius, but at 37 degrees celsius.

6.  All 5 of the secretory mutant candidates secreted nearly normal levels of invertase at 24 degrees
celsius, but at 37 degrees celsius showed a greater difference between the rates of protein synthesis
and invertase secretion.
 Unlike the class a sec. mutants, the new mutants did not accumulate derepressed invertase
activity.
 However, cytoplasmic invertase, but not secretory invertase was synthesized (class b secretory
mutants)

8.  Aided by the use of a high copy number plasmid that contains the invertase gene (suc2) on an
insert.
 Mutant and wild-type transformed cells derepressed and radiolabeled with (so4)2- for 30
minutes.
 Secreted proteins are released and accumulated proteins are retained within the spheroplast.

10.  Electrophoretic mobility of accumulated invertase precursors suggested partial glycosylation.
 binding to cona-sepharose and sensitivity to endo h
 Mutant and wild-type cells labeling
 insoluble and soluble fractions
 Protein bounding separated by sedimentation
 Comparison with unbound/untreated fractions after immunoprecipitation of invertase

12.  Linking of oligosaccharides to yeast mannoproteins
 63-kalton form produced when invertase from wild-type cells was treated with endo h
 Represents a 60-kdalton polypeptide with 9-10 remaining Glcnac residues
 Endo h-resistant persisted in sec 59 invertase
 Endo h treatment effect
 Sec 53 had little carbohydrate, while 2-3 of sec 59 invertase were glycosylated.

14.  Treatment of wild-type cells with tunicamycin
 incorporation of [^3h] mannose
 Inhibition of protein synthesis with cycloheximide
 Mannose transport not affected
 Secretory mutants secreted normally at 24 degrees celsius, but showed a dramatic reduction at 37
degrees celsius.
 Sulfate permease derepression not affected by treatment of wild-type cells with tunicamycin

16.  Treated cells converted to spheroplasts
 Tnp-tagged proteins immunoprecipitated
 Wild-type cells at 37 degrees celsius and 25 degrees celsius, and secretory mutant cells at 25
degrees celsius, secreted the same set of surface proteins.
 Permissive incubation conditions
 Subsequent tnbs treatment allows tagging of intracellular proteins that are released when cells
are converted to spheroplasts.

18.  Localization of cpy requires part of the secretory pathway
 Class a secretory mutants block cpy transport
 Immunoprecipitation with affinity-purified antibody directed against cpy
 Unglycosylated cpy produced in sec 53 and sec 59(37 degrees celsius); mature cpy produced at 24
degrees celsius
 59-kdalton form produced(wild type/pep4 cells treated with tunicamycin) at 37 degrees celsius
 In the absence of tunicamycin, they produced the expected 61 and 69-kdalton
 Conditions only delay the transport of unglycosylated cpy to the vacuole

20.  Er golgi body vesicle  cell surface
 Mutations affecting early stages are epistatic to mutations that block later stages
 If sec 53 or 59 blocks a step before the one blocked in sec 18, then double sec mutants should fail
to accumulate active invertase at 37 degrees celsius.
 Much less invertase was accumulated in the double mutants than in only sec 18.
 Sec 53 and sec 59 are dependent on sec 18.

21.  assessing the cytologic consequences of the lesions
 When transport from the er is blocked in class a sec mutant, the er tubules and nuclear envelope
expand…
 Sec 59 showed fragmented er tubules of variable width
 Mutant cells and wild-type cells treated with tunicamycin at 25 degrees celsius were normal
 Sec 53 not as altered as sec 59; rounded vesicles in place of thin er tubules.

23.  New yeast secretory mutants are identified that are defective in the
processing and transport of exported proteins.
 The Class B secretory mutants were found to be temperature-sensitive
for the production of active secretory forms of invertase and acid
phosphatase, but not for the synthesis of active cytoplasmic invertase.

24.  Inhibition of maturation and transport of secretory, plasma
membrane, and vacuolar enzymes in sec 53 and sec 59 indentified a
common step in the translocation of polypeptides across the ER
membrane in yeast, similar to analogous proteins in mammalian
cells.