2001 Summer Scholar Participants-HHMI Undergraduate Program - Carnegie Mellon University

2001 Summer HHMI-supported Participants

Emily Drill

Emily Drill, Carnegie Mellon University
(Mentor: Dr. Elizabeth Jones)

The Role of PBN1 in the Unfolded Protein Response Pathway

PBN1 is an essential gene found in Saccharomyces cerevisiae. The Pbn1 protein is an integral membrane protein of the endoplasmic reticulum, and is involved in post-translational processing of the PrB (protease B) precursor. Preliminary data suggests that PBN1 may be required for the unfolded protein response (UPR) pathway. The goal of this project is to try to determine the essential function of PBN1.

In yeast, accumulation of unfolded proteins in the ER triggers the UPR pathway, resulting in the upregulation of chaperones required for protein folding. This accumulation triggers the dimerization of Ire1p (an ER integral membrane kinase), which then transautophosphorylates. The cytosolic domain of Ire1p splices the intron out of the HAC1 mRNA, resulting in the expression of the Hac1p. Hac1p is a transcription factor that binds to the unfolded protein response element (UPRE) in the promoter regions of known chaperone genes such as KAR2, upregulating them.

The reporter construct UPRE-lacZ was transformed into wild-type PBN1 and mutant pbn1-1 strains so that the level of lacZ expression of these cells, which can be measured by β-galactosidase assay, is directly proportional to UPR activity. Assays of untreated cells showed lower lacZ expression in the mutant strain than in the wild-type, suggesting that PBN1 is involved in the UPR. UPR activity was also determined for cells treated with the drugs tunicamycin and DTT, both know to increase the levels of unfolded proteins in the ER. Assays of tunicamycin-treated cells showed a significant increase in lacZ expression over time in both strains, but the mutant always stayed lower than the wild type. Drug sensitivity testing showed that the mutant is actually more resistant to tunicamycin than the wild type. Further testing will be done to determine whether this resistance co-segregates with the pbn1-1 mutation.

In addition, PBN1 is being tested for genetic interaction with and ERO1, IRE1, HAC1, and PDI1. Diploid strains were created which contain pbn1-1 and either ero1-2, ire1::KANMX4, hac1::KANMX4, or pdi1CSCCCS. If the haploid strains containing these double mutations fail to grow, it suggests that the double mutants are synthetically lethal and there is interaction. All four spores from the dissection of the pbn1-1/ero1-2 double mutant strain grow under normal conditions, but further testing may show differences in growth between the double mutant spore and the other spores. The three other diploids have not yet been tested.

Christian Fidler

Christian Fidler, Carnegie Mellon University
(Mentor: Dr. Elizabeth Jones)

The Effect of Mutation in the Zinc Finger Domain of the PEP5 gene on the Binding Interactions of Pep5p with Other Vacuolar Trafficking Proteins in Saccharomyces cerevisiae

PEP5 has been previously found to encode a protein responsible for both biogenesis and maintenance of the vacuole in Saccharomyces cerevisiae. In addition, it is believed that the product of the PEP5 gene functions in at least two trafficking steps during transport of the hydrolases, Protease A (PrA), Protease B (PrB), and Carboxypeptidase Y (CpY). Specifically, Pep5p functions in trafficking between the Golgi to Endosome step as well as in the Endosome to Vacuole step. Previously, the presence of Pep5p function at each of these steps was characterized through identification of genetic interactions between PEP5 and other genes known to be involved at each of the aforementioned steps. As one example, a particular mutant allele of the PEP5 gene (PEP5 :: TRP1) was found to genetically interact with a mutant allele of the Vps8 gene (Vps8-200) whose protein product is known to function in vesicle docking and fusion as well as in recycling between the Golgi and Endosome (Woolford et al, 1998). Consequently, the physical nature of the modes by which these proteins interact is of great interest if more is to be learned about the function of Pep5p in vacuolar transport. It is currently suspected that the zinc finger domain of Pep5p may be responsible for the observed protein-protein interactions in which Pep5p is involved. As a result of this suspicion, the goal of this summer's work is to test whether mutations in the zinc finger domain of Pep5p abolish the interaction between Pep5p and other proteins with which Pep5p is known to interact. This task will be accomplished by analyzing several zinc finger mutants of the PEP5 gene. Each pep5 zinc finger mutant isolated will be sub-cloned into the yeast two hybrid system using the technique of gap repair, and will be tested for the ability to interact with other known vacuolar trafficking proteins to ascertain if the zinc finger region is responsible for the observed interactions. To this point in the project, the fourteen different pep5 zinc finger mutant alleles have been assessed phenotypically, using an assay to detect the presence of functional Carboxypeptidase Y, and sub-cloned into vectors for use in the yeast two hybrid system.

Theresa Nguyen

Theresa Nguyen, Carnegie Mellon University
(Mentor: Dr. John Woolford)

Analysis of Novel Proteins Present in Pre-Ribosomal Particles

Ribosome assembly is an essential process that occurs in the nucleolous of all eukaryotic cells. Ribosomes are necessary for protein synthesis, and without them, survival would not be possible. During the course of ribosome assembly, RNA processing takes place, and many proteins are involved in the formation of the final product. However, not all of the same proteins are found in both the 40s and 60s ribosomal subunits, and they are not always present throughout each step of the assembly pathway. Additional research is needed on the exact role of the proteins during ribosome synthesis, and where exactly they are found in the cell.

The main goal of this project was to analyze proteins that have been identified in pre-ribosomal particles and then trace them through the cell to better understand their function in ribosome assembly. In particular, the YGL111W, YER126C, Rrp1, Erb1, and Rlp24 genes of Saccharomyces cerevisiae were examined. The proteins were epitope-tagged by introducing a corresponding DNA sequence into the gene through transformation and recombination. This DNA sequence included a selectable marker for histidine as well as a hemagglutinin tag, and was obtained through a series of PCR reactions. The sequence was then incorporated into the yeast genome by genetic recombination through transformation. Protein extracts were taken from the transformed yeast strains and separated by SDS-PAGE gel electrophoresis. The proteins were then transferred to a Western blot filter and probed with the specific antibody for the HA-tag. The results of this test allowed for detection of the properly tagged proteins. More detailed analysis of the proteins was done by sedimentation on sucrose gradients followed by fractionation in order to determine the localization of the proteins. Future studies involving immunofluorescence can hopefully give more insight into the exact function and subcellular location of the proteins in the ribosomal assembly pathway.

Traci Prouser

Traci Prouser, Cedar Crest College
(Mentor: Dr. Charles Ettensohn)

Dynamics of GSK-3 beta Turnover in Lytechinus variegatus Studied using Time Lapse Microscopy and Image Processing

Glycogen synthase kinase (GSK-3 beta), a component of the Wnt signaling pathway, is known to control the position of the boundary between the ectoderm and the endoderm territories and thus mediate patterning along the animal-vegetal (AV) axis in the sea urchin. It also functions in degrading beta-catenin, another molecule in the Wnt signaling pathway responsible for the activation of target genes. It has been previously shown in this laboratory that beta-catenin is degraded more rapidly in cells of the animal pole than in cells of the vegetal pole. It is our intention to test whether this is a direct result of the differential degradation of GSK-3 beta on the vegetal side of the embryo. Fertilized sea urchin eggs will be injected with mRNA encoding GFP-tagged GSK-3 beta. After accumulation of the protein, translation will be blocked with emetine. With the aid of time lapse microscopy, the level of fluorescence will be quantified and analyzed with image processing software.

Matthew Wood

Matthew Wood, Carnegie Mellon University
(Mentor: Dr. Elizabeth Jones)

Determination of the interacting domain of the mature region of Protease B in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the vacuolar protein Protease B (PrB) precursor undergoes a number of modifications in the endoplasmic reticulum before it is sent to the vacuole. First the N-terminal signal sequence is cleaved off, then the protein undergoes an autocatalytic internal cleavage that divides the precursor into a propeptide and a mature region. Two-hybrid studies in the Dr. Jones lab have shown that these two regions interact with each other, though the mechanism of this interaction is unknown. Previous studies in the lab have indicated that deleting the coiled-coil region of the propeptide disrupts the interaction. The goal of this study has been to identify what region(s) of the mature peptide are required for this interaction to take place.

To identify the interacting regions, a series of systematic prb deletions were created with custom PCR primers. These primers were designed to have sequence homology upstream of the PRB mature region and at some distance into the gene itself, but not in the region in between. The product of the PCR reaction has some part of the mature sequence deleted. Two-hybrid analysis was used to determine if the deleted region is required for the interaction to take place. Four different prb deletions were created, with deletions of 360, 600, 720, and 900 nucleotides from the mature region coding sequence. With wild type pro and mature regions, the interaction in two hybrid assay should allow colonies to grow at a concentration of at least 25 mM 3AT. It was found that all of the available prb deletions were not viable above 10 mM 3AT. This suggests that the critical binding region in the mature propeptide is coded somewhere in the first 360 base pairs of the sequence. Future studies will be aimed at identifying the type of binding that is occurring between the two regions.