BISC220/S12: Mod 2 Background

Lab 6: Phenotypes of Secretory Mutants Lab 7: Analyzing Secretion Defects by Western Blotting Lab 8: Probing and Detecting the Western Blot Media Recipes

Phenotypes of Secretory Mutants
Yeast as a Cell Biological Tool Our current understanding of many important cell biological processes is based on a combination of biochemical, microscopic, and genetic studies. The unicellular eukaryote Saccharomyces cerevisiae, often referred to as budding yeast, is an important model organism that can be studied by all three of these approaches, but it is an especially valuable tool for genetic studies of cell biology. Through the execution of elegant genetic screens, many yeast mutants that are defective in particular cell biological pathways were isolated. Characterization of such mutants and the cloning of the responsible genes led to the identification of important components and mechanisms of many cellular processes, including the cell division cycle and protein sorting and secretion. Because many of these processes have been highly conserved throughout eukaryotic evolution, similar factors and mechanisms discovered in yeast still exist and function in “higher” eukaryotic cells such as mammalian and human cells. Therefore, studies in the simple, genetically-amenable yeast S. cerevisiae have tremendous value in working out complex cell biological processes that function throughout the eukaryotic lineage. Yeast nomenclature: In yeast, the wildtype allele of a gene is written in upper case italics and is followed by a number (e.g. SEC61). A mutant version of that gene is written in lower case italics (e.g. sec61). The protein product encoded by a gene is written Sec61 or Sec61p. Protein Targeting & the Secretory Pathway For the next three weeks, we will focus our investigative efforts in the laboratory toward an important topic in cell biology: the process by which proteins are sorted to their proper destination compartments within eukaryotic cells. In particular, we will examine protein targeting to the endoplasmic reticulum (ER) and through the secretory pathway (Golgi apparatus, lysosomes, vacuole, etc.) in S. cerevisiae. This entire process is often termed protein secretion, even though not every “secreted” protein is actually expelled from the cell. You should read some background information on protein secretion in your text to familiarize yourself with some of the details of the secretory pathway. A basic overview of protein secretion is provided in Figure 1 below. Figure 1. A simple overview of protein secretion in eukaryotic cells, including yeast. This process is broken down into four general steps as indicated. The large arrow shows the overall flow of proteins through the secretory pathway. To study protein secretion in yeast, we will work with wildtype (WT) and two secretion defective (sec) mutant yeast strains. Given the importance of protein sorting to the ER and through the secretory pathway, a complete block in this process should be lethal to the cell. So the question arises: How can we study yeast that are defective for protein sorting and secretion if they are dead?! The answer lies in the fact that sec mutants we will use are not always fully blocked for protein secretion. Instead, these are “conditional mutants” – mutants that are blocked for secretion only under certain conditions, in this case at high temperatures. At room temperature (25˚C), the sec mutants grow reasonably well and display only a moderate block in protein secretion, if any. We refer to room temperature as the “permissive temperature” for these strains because it permits them to grow relatively normally. At 30°C, however, (a “semi-permissive” temperature), their protein secretion defects become more pronounced and they may not grow as well. Finally, at 37˚C (the “non-permissive” temperature), they lose viability almost completely. On the basis of the experiments that we will perform in lab, as well as on background research using an online resource, the Saccharomyces Genome Database, you will be able to hypothesize which step of the secretory pathway is blocked in the two secretion-defective mutants. Overview of the Experiments Your investigation of the yeast sec mutants will involve two types of experiments. First, you will try to confirm which step of the secretory pathway is blocked in each of the two mutant strains by making use of a specially designed reporter gene. This reporter gene is described in the bottom of Panel 12-1 on p. 703 in Molecular Biology of the Cell, 5th ed. (Alberts, 2008). In the first lab of this series you will set up the reporter gene assay by spotting a number of transformed yeast strains onto various types of growth media. Since the yeast will require about a week to form visible colonies, you will analyze your results in Lab 2. In addition, you will be given the names of the genes that are mutated in the two strains; as part of your homework assignment that will be due in Lab 2, you will be asked to search the Saccharomyces Genome Database (SGD) and to consult some research articles to summarize the functions of the proteins that are encoded by these genes. You will use this knowledge to predict the growth patterns in the reporter gene assay, and later you will compare your predictions with your experimental results. You will also perform BLAST searches to identify homologs of the two yeast secretory proteins that may exist in other species. Through this exercise, you will gain a sense of the evolutionary conservation of these proteins. In the second part of the lab series, you will perform a Western blot (also known as an immunoblot) to observe the forms of a normally secreted protein that accumulate in the two sec mutants. The experimental procedures for the Western blot will be split between Labs 2 and 3. At the end of the series, you will be asked to describe your investigations in a formal scientific paper.

Yeast Strains
Three yeast strains will be employed in our laboratory work. All are auxotrophic mutants, meaning that each is defective in the production of at least one essential nutrient:
 * 1) WT: This strain is wildtype for protein sorting through the secretory pathway (SEC+).  It does, however, contain a number of chromosomal mutations, including mutations in the URA3 and HIS4 genes, which are required for biosynthesis of the ribonucleotide uracil and the amino acid histidine, respectively.  As a result, this strain is unable to grow on medium that does not contain uracil and histidine. In proper yeast genetic notation, the relevant genotype of this strain is:  ura3 his4. You should include all strain genotypes using proper nomenclature in the Materials & Methods section of your paper.
 * 2) sec18: This strain is identical to the WT strain, except that it contains a temperature sensitive mutation in the gene SEC18.  As a result, this strain dies at high temperatures (37˚C) and is unable to efficiently secrete proteins (i.e. displays a sec phenotype) due to a defect in a specific step in the secretory pathway. The genotype of this strain is: ura3 his4 sec18-1.
 * 3) sec61: This strain is also identical to the WT strain, except that it contains temperature sensitive mutation in the gene SEC61. This mutation causes a defect in the secretory pathway at a step that is different from the step that is affected by the sec18 mutation. The genotype of this strain is: ura3 his4 sec61-1.

Plasmids
In Lab 1, you will be given plates with colonies of WT, sec18 and sec61 yeast that have been transformed with one of three plasmids you will use for the genetic reporter assay (see below). The term “transformation” means that those cells have successfully taken up the desired plasmid DNA into their nuclei and are expressing the genes encoded by that DNA. The transformed cells are referred to as “transformants.” Whenever the transformants replicate their genomic DNA, they also copy the plasmid DNA; however, the plasmid copies are not as faithfully segregated between the two daughter cells as the yeast chromosomes. Therefore, we must use a trick called “genetic selection” to ensure that all the yeast cells we are going to test are really transformants. All three of the plasmids you will use for the reporter assay contain the wildtype URA3 gene (as well as other sequences), which functions as a “marker” for the genetic selection: because all our yeast strains have a mutation in their genomic copy of the URA3 gene (i.e. their genotype for this locus is ura3), only the cells that have acquired the reporter plasmid during transformation and retain it during cell division will be able to grow on growth medium that lacks uracil. Throughout the lab you will be using such ura- medium, so you can be sure that all the yeast cells growing on the plates contain the appropriate plasmids. You will be given plates of WT, sec18 and sec61 yeast cells that have been transformed with plasmids called YEp24, pRSB203, and pRSB204 (a total of nine transformed strains). The YEp24 and pRSB203 plasmids will serve as controls, whereas pRSB204 contains the reporter gene, signal sequence tagged HIS4 (ss-HIS4). This reporter will allow you to identify mutants with a particular defect in the secretory pathway. The features of the three plasmids that are relevant to our study are described below and depicted in Figure 2. The reporter assay employs genetic selection based on histidine and the HIS4 gene in addition to the URA3-based selection used to retain the plasmids. 'Do you think wild type SEC yeast (with defective chromosomal genes his4 and ura3'') transformed with a plasmid encoding a chimeric ss-His4 protein will grow on medium lacking histidine? Remember that histidine is required in the cytoplasm of the cell.''' Figure 2. Composition of the YEp24, pRSB203, and pRSB204 plasmids. All three contain the marker gene URA3. pRSB203 contains the normal HIS4 gene with a linker sequence at its 5’ end (the linker is translated, but does not affect the function of the His4 protein). pRSB204 contains ss-HIS4, which encodes a His4 protein that contains an N-terminal ER signal sequence. The signal sequence is separated from the rest of the His4 protein by the same linker contained in pRSB203. Using the Reporter Gene to Investigate Defects in the Secretory Pathway In Lab 5, you will test the ability of the nine transformed yeast strains (WT, sec18, and sec61 strains, each transformed with YEp24, RSB203, and RSB204) to grow on two types of media plates (ura- and ura-/his-) at three different temperatures (25°, 30°, and 37°C). One week of incubation will be required for the phenotypes to be assessed accurately. You will use that time to do some research to learn about the functions of the SEC18 and SEC61 gene products and, based on what you learn, predict the growth phenotypes of all nine strains under the various conditions. In Lab 6, you will compare your predictions to your actual results and develop hypotheses about the where the blockage in the secretory pathway might be to explain the growth patterns that you observe. The Western blot that you will perform in Labs 6 & 7 will allow you to test your hypotheses. References Deshaies, R.J. and Schekman, R. (1987). A Yeast Mutant Defective at an Early Stage in Import of Secretory Protein Precursors into the Endoplasmic Reticulum. J. Cell Biol. 105:633-645. (available online at http://www.jcb.org/) Novick, P., Field, C., and Schekman, R. (1980). Identification of 23 Complementation Groups Required for Post-translational Events in the Yeast Secretory Pathway. Cell 21:205-215. (posted on lab e-reserve conference) Novick, P. Ferro, S., and Schekman, R. (1981). Order of Events in the Yeast Secretory Pathway. Cell 25:461-469. (posted on lab e-reserve conference)
 * 1) YEp24: This is the plasmid backbone of pRSB203 and pRSB204. It contains the URA3 gene, but no other genes that will be expressed in yeast. Strains transformed with this plasmid will serve as negative controls in our experiments since this plasmid contains no form of the HIS4 gene. Strains transformed with YEp24 should not be able to grow on media lacking histidine.
 * 2) pRSB203: This plasmid has the HIS4 gene inserted into YEp24. It also contains the wildtype URA3 gene.  HIS4 encodes histidinol dehydrogenase, a cytoplasmic enzyme that catalyzes the last step in the biosynthesis of the amino acid histidine, which is the conversion of histidinol to histidine. The yeast strains we will be using cannot normally grow on medium that lacks histidine, since they have a mutation in their genomic copy of HIS4, but the plasmid-borne copy of HIS4 will allow them to produce functional His4 enzyme and therefore to grow on his- medium. The strains transformed with pRSB203 will therefore serve as positive controls in our experiments.
 * 3) pRSB204: This plasmid contains a chimeric gene, which consists of the HIS4 gene with an additional stretch of DNA that encodes a signal sequence fused to its 5’ end (ss-HIS4). The term “chimera” refers to something that is composed of parts that come from different sources. A signal sequence is a short peptide sequence composed mainly of hydrophobic amino acids that targets a protein to the ER during the first step of protein secretion (see Figure 1). The ss-His4p (fusion protein) will therefore enter the ER and travel through the secretory pathway, in constrast to the normal His4 protein, which remains in the cytoplasm.