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

Saccharomyces cerevisiae S. cerevisiae in light microscopy & SEM

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/)

Major Players Peter Novick Randy Schekman at UC Berkeley

Example of a sec mutant: sec1-1 WT 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

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

Using a reporter gene to study secretion

A portion of the histidine biosynthethic pathway

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)

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

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

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?

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.

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.