1. BISC 220 Lab—Series 2 Protein Transport through the Secretory Pathway
1. A Genetic Assay
Learning about the normal process by studying yeast mutants
2. Western Blot
- Following a secreted protein’s progress through the pathway in WT and mutants

2. Saccharomyces cerevisiae
S. cerevisiae in light microscopy & SEM

3. Why Yeast? Simple eukaryote Genome sequenced
Genetic engineering creates useful “designer” strains carrying “reporter genes”
Cheap, easy to grow, short generation time, easy to work with
Genetically tractable
Can easily make mutants, combine several mutations in one strain, identify genes responsible for mutant phenotypes, etc.
Yeast can exist in both haploid and diploid states (2 mating types: MATa & MAT; diploid = a/)

5. Major Players
Peter Novick Randy Schekman
at UC Berkeley

6. Example of a sec mutant: sec1-1WT
sec1-1
Ways to identify sec mutants:
Isolate denser cells by spinning through density gradient (e.g. Novick et al., 1980)
Select/screen for cells that fail to secrete some specific protein (e.g. Novick et al., 1981; Deshaies & Scheckman, 1987)
Use of conditional (temperature-sensitive) mutants to study defects in essential genes

7. Behavior of temperature-sensitive mutants
Permissive Temperature
(e.g. 25°C, room temp.)
Protein produced from mutant gene functions normally or close to normally; yeast grow fine & don’t have a strong abnormal phenotype
Non-permissive Temperature
(e.g. 37°C)
Protein produced from mutant gene does not function; yeast cannot grow & exhibit an abnormal phenotype when shifted to the NPT for a short time
Semi-permissive temp.—between PT & NPT; yeast exhibit partial abnormal phenotype

8. Using a reporter gene to study secretion

9. A portion of the histidine biosynthethic pathway

10. Yeast Strains You Will Use
sec61 & sec18
Different defects in the secretory pathway
temperature sensitive (ts)
RT is permissive temperature -”normal” SEC pathway
37°C is non-permissive - SEC blocked (lethal)
30°C is semi-permissive; should see partial mutant phenotype
Wild Type (WT)
Normal SEC genes; not temperature sensitive (ts)
All 3 have auxotrophies:
ura3- can’t make uracil
his4-can’t make histidinol dehydrogenase (the enzyme that breaks down histidinol, the precursor to histidine)

11. Reporter Gene & Control Plasmid Constructs
Plasmids constructed from YEp24 backbone
URA3 allows selection of transformed cells on selective media
pRSB203
URA3
HIS4 encodes normal protein used to make histidine IN THE CYTOPLASM
pRSB204
ss-HIS4 encodes a fusion protein targeted to ER so takes HIS4 product OUT OF CYTOPLASM

12. Growth on Selective Media: Things to Consider
Temperature
RT permissive—normal SEC function
30°C borderline—mutant phenotype shows but not lethal (may be different for two strains)
37°C - lethal to both sec mutants
SD -U
Lacks Uracil but contains Histidine
Selects for plasmid-containing cells
SD-U-H
Lacks both Uracil and Histidine but contains histidinol, the substrate for the product of HIS4 gene required in the pathway to making histidine
Differentiates cells expressing ss-His4 fusion protein from those expressing His4

13. Experimental Question: What is the role of Sec61p and Sec18p in the secretory pathway in yeast? Or What are the specific defects of the sec61-1 and sec18-1 mutants?

14. To Do Today
Spot nine yeast strains onto two types of media at three different temperatures (6 plates total); let grow until next week.
Research the functions of the Sec61p and Sec18p proteins using the Saccharomyces Genome Database.
Perform BLAST analysis with Sec61p and Sec18p protein sequences.
After you understand the functions of the two proteins, fill in the yeast growth prediction chart.

15. Assignment Due Next Lab
Completed yeast growth prediction chart
Paragraph summarizing what you have learned about Sec61p and Sec18p from the SGD search and BLAST analysis and explaining how you used that knowledge to make your growth predictions.