BISC220/S12: Mod 2 Lab 6: Difference between revisions
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[[BISC220/S12: Mod 2 Background | Background Information]]<br> | [[BISC220/S12: Mod 2 Background | Background Information]]<br> | ||
[[BISC220/S12: Mod 2 Lab | [[BISC220/S12: Mod 2 Lab 6 | Lab 6: Analyzing Secretion Defects by Western Blotting]]<br> | ||
[[BISC220/S12: Mod 2 Lab | [[BISC220/S12: Mod 2 Lab 7 | Lab 7: Probing and Detecting the Western Blot]]<br> | ||
[[BISC220/S12: Mod 2 Media Recipes | Media Recipes]]<br> | [[BISC220/S12: Mod 2 Media Recipes | Media Recipes]]<br> | ||
Revision as of 11:19, 23 February 2012
Background Information
Lab 6: Analyzing Secretion Defects by Western Blotting
Lab 7: Probing and Detecting the Western Blot
Media Recipes
Analyzing Secretion Defects by Western Blotting
In today’s lab you will have two main tasks:
- Scoring the results of the ss-HIS4 reporter gene assay
- Performing the initial steps of a Western blot, using a different reporter, to determine which forms of a normally secreted protein accumulate in the two sec mutant strains
Protein Maturation in the Secretory Pathway
Proteins that travel through the secretory pathway usually undergo several types of modifications before they reach their mature forms. The signal sequence that targets secreted proteins for co-translational insertion into the ER is usually cleaved off by a signal peptidase just after that part of the protein enters the ER, even before the entire protein has been synthesized by the ribosome (Waters, 1988). Second, most secreted and plasma membrane proteins are modified by the addition of carbohydrates in a process called glycosylation. Branched chains of sugar molecules (oligosaccharides) are initially added to proteins in the ER in a process called glycosylation. Some trimming of these chains occurs before proteins leave the ER and proceed to the Golgi. In the Golgi, additional carbohydrate modifications often occur. As a result, multiple, differently glycosylated forms of a particular secreted or cell surface protein are present in a cell at any one time, as different molecules of the protein travel through the secretory pathway. Since they have different molecular weights, these various forms of a protein can be separated by SDS-PAGE and, if an antibody is available, can be detected by Western blotting.
Pre-pro-α-factor
In this experiment you will perform a Western blot to detect the various forms of a normally secreted protein, α-factor, that are present in the wildtype and sec mutant yeast cells. Alpha-factor is a yeast mating pheromone. Yeast can exist as both haploid and diploid cells. Haploid yeast come in two mating types (MAT): a and α. MATa yeast secrete a pheromone called a-factor, while MATα cells secrete α-factor. These pheromones allow yeast cells to sense cells of the opposite mating type that are in close proximity and to initiate events that prepare them to mate (i.e. to fuse into a diploid cell). We will talk more about yeast mating factors in the cell signaling portion of the course, but for now, the important thing is that they are secreted proteins that must travel through the ER, the Golgi, and subsequent compartments before being released at the cell surface by exocytosis.
The mature, secreted form of α-factor is a polypeptide of only 13 amino acids, but it is synthesized as a larger precursor protein that is processed to its mature form in several glycosylation and proteolysis steps. Using the nomenclature that is generally applied to secreted polypeptides derived from larger precursors (e.g. many hormones), the form of α-factor that is translated by the ribosome is referred to as “pre-pro-α-factor” (pp-αF). This form includes the ER signal sequence and other “spacer” sequences that are eventually removed to generate the mature α-factor polypeptide. The term “pro-α-factor” (minus the “pre-“) would typically refer to an intermediate form of the final product, after cleavage of the signal sequence (which usually occurs just after a protein’s translocation into the ER) but prior to removal of spacer sequences.
An antibody raised against pre-pro-α−factor would be expected to recognize all forms of the protein as it progresses through the various glycosylation and proteolysis steps in its processing sequence. The molecular weight of the unglycosylated, translated form (including the signal sequence) is approximately 18.6 kDa. In the ER, N-linked core oligosaccharides are added to three sites in the protein, causing its molecular weight to increase significantly; the fully modified ER form of pp-αF is 26 kDa (Julius, 1984). Many secreted proteins undergo additional carbohydrate modifications in the Golgi, but glycosylation of pp-αF is completed by the time it leaves the ER. As pp-αF moves through the Golgi and into the secretory vesicles that bud from the Golgi, endo- and exo-proteolytic steps eventually produce the mature, secreted form of α-factor, reducing it to 3.4 kDa (see Fig. 1).
Figure 1. Processing of pre-pro-α-factor as it passes through the secretory pathway (Julius, 1984).
Using pre-pro-α-factor to investigate the steps blocked in sec18 and sec61 mutant yeast
For the Western blot analysis you will probe for precursor forms of α-factor in WT, sec18, and sec61 yeast cells that were either grown continuously at room temperature (RT, 25°C) or grown at RT then shifted to 37°C for one hour. Note that all of these strains are haploids of mating type alpha (α) (MATα): If they were mating type a (MATa) haploids or if they were diploid strains, they would not produce α-factor! Recall that the sec18 and sec61 strain each have a temperature sensitive defect in a different step in the secretory pathway. The Western blot will allow you to determine the molecular weight of the form of α-factor that accumulates in each of these strains due to the block in secretion. The WT strain will serve as a comparison control and a negative control lysate of a wildtype MATa strain that doesn’t express any alpha factor will be supplied.
What should happen to α-factor in a cell expressing it that has a normal secretory system?
References
Julius, D., Schekman, P., and Thorner, J. (1984). Glycosylation and Processing of Prepro-α-Factor through the Yeast Secretory Pathway. Cell 36:309-318. (posted on lab e-reserve conference)
Karp, G. (2005). Cytoplasmic Membrane Systems: Structure, Function, and Membrane Trafficking. In: Cell and Molecular Biology: Concepts and Experiments, 4th ed. John Wiley & Sons, Inc., Hoboken, NJ. p. 279-333.
Waters, M.G., Evans E.A, Blobel, G. (1988) Prepro α factor has a cleavable signal sequence. Journal of Biological Chemistry 263(13): 6209-6214.
Assignment
Assignment 5: Yeast Growth Predictions/BLAST
This should be started during lab time so you can ask any questions you might have.