Yeast Secretory mutants that block the formation of active

cell surface enzymesby: susan Ferro- Novick, peter novick, charles field, and

randy schekman

Secretory pathway

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.

introduction

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).

Materials and methods

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

density enrichment procedure

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.

Density enrichment procedure con…

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)

Immunoreactive invertase accumulated in sec 53 and sec 59

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.

Immunoreactive invertase accumulated in sec 53 and sec 59 con…

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

Immunoreactive invertase accumulated in sec 53 and sec 59 con…

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.

Other cell surface- related defects in sec 53 and sec 59

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

Other cell surface- related defects in sec 53 and sec 59 con…

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.

Influence on vacuolar glycoprotein synthesis

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

Mutations block early in the secretory pathway

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.

Mutations block early in the secretory pathway

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.

conclusion

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.

significance

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.

I hope you all learned something!the end.