My Favorite Yeast Protein(s):

Ste2 (annotated) and YFL030W (unannotated)

This web page was produced as an assignment for an undergraduate course at Davidson College.

My Favorite Annotated Yeast Gene's Protein: Ste2

Many different databases can reveal pertinent information about a particular gene product. This web page will go through several of these websites dealing with the protein associated with the yeast gene Ste2, discussing both the nature of the alpha-pheromone receptor encoded by it and this protein's place in the overall nature of the yeast cell.

DIP

The DIP (Database of Interacting Proteins) website is useful in finding relationships between gene products, both real and theoretical. A search in this website for the gene Ste2 yielded relatively uninteresting results: there were three genes that the researchers thought interacted with it, but all three were hypothetical proteins (YHR016c, YGR141w, and YOR242c) and the degree of certainty of their connection wasn't very strong.

A map of the interactions between protein Ste2 (in red) and other gene products. The cluster above yielded little interesting findings. Courtesy DIP.

Yale Gerstein Lab

A search of Ste2 at this website tells us some of what was already known about Ste2; it is found in the membrane of yeast cells and is not essential for cell viability. This website also gave some interesting information about expression levels, however.

Courtesy Yale Gerstein Lab.

This description of the Ste2 protein gives us absolute and relative levels of expression which may be used in comparison with other proteins. Again, it confirms what was found in the microarray data -- Ste2 is especially expressed in great amounts in the presence of alpha factor.

TRIPLES Database

The TRIPLES website can be used to search for clones in which the gene in question is disturbed by a mTn transposon. The results of experiments can then give clues as to the nature of your gene and its product. Unfortunately, no results were found for Ste2.

Munich Information Center for Protein Sequences

The data from the Munich Information Center for Protein Sequences database confirms earlier knowledge about Ste2. Indeed, we find from this website, which utilizes information from SGD and PubMed, that internalization of the protein is not important for mating response, that the receptors localize to the tip of the mating projection, that it is delivered into the vacuole lumen by transport vesicles, and that the receptor is not recycled after internalization.

PDF Files on Protein Connections

Searches for Ste2 from the following files yielded no matches: Aging (PDF format), Membrane (PDF format), and Degradation (PDF format).

YRC Two-Hybrid Analysis

The University of Washington's Yeast Resource Center's two-hybrid analysis yielded no hits of Ste2.

WIT

There was no information of use relating to Ste2 in the What is There? website.

Experiments/Conclusions

The gene product of Ste2 is so well documented that, unless it is found to interact with other proteins or gene products in new ways, most of the searches contain redundant material. Unfortunately, the new studies done with protein arrays and the like have not had a tremendous impact towards the alpha pheromone receptor encoded by Ste2. For this reason, I am hesitant in stating that novel experiments could do a tremendous amount to further our knowledge of this proteins function. We know that it is necessary for yeast sexual reproduction and that it is not critical for viability. This gene product is involved in so much of yeast biology that new experiments seem tough to develop. However, even though this study seems cut and dried, there may be some hidden pathway that could be uncovered in years to come.

My Favorite Unannotated Yeast Gene's Protein: YFL030W

Another important aspect of web searches is their ability to explain relationships between an unknown protein and the characterized proteins. By doing searches and examining the proteins related to the gene product of YFL030W, we can further determine its nature and test some of our earlier predictions.

DIP

The DIP (Database of Interacting Proteins) website is especially useful in this search, as it gives a starting place for looking at some of the preliminary finding on YFL030W and by examining those proteins that interact with the annotated gene. This gene is categorized by SwissProt as a class-V of pyroxidal-phosphate-dependent aminotransferases with a pyridoxal phosphate cofactor. Four proteins matched the search, including two protein kinases of note, PHO85 and DUN1. DUN1 is a transducer of the DNA damage signal while PHO85 is activated when phosphate levels are high, and it phosphorylates the PHO4 transcription factor which creates repression. Although this initially might put YFL030W in this group, the confidence the researchers put in this were fairly weak, so these might not be 'true' interactions.

A map of the interactions between protein YFL030W (in red) and other gene products. Courtesy DIP.

Yale Gerstein Lab

A search of YFL030W at this website had no information on this protein, and merely confirmed what was already known through previous searches

TRIPLES Database

The TRIPLES website can be used to search for clones in which the gene in question is disturbed by a mTn transposon. The results of experiments can then give clues as to the nature of your gene and its product. Many hits were found using this website, and all of the mTn transposon insertions yielded a 385 aa protein. Most of the clones found yielded the same results.

These results indicate that, for this particular clone, YFL030W is found throughout the cell stages, and the LacZ expression level indicates that this protein was being expressed in the cell when the mTn transposition took place. Additionally, the 'HapTra' Assay is designed to show "cell inviability of haploid transformants" which may indicate that this protein is necessary for immediate cell function. The '10Ben' assay shows that there is a Benomyl hypersensitivity when this gene is disrupted, though the exact importance of this is not immediately obvious.

Courtesy TRIPLES.

Munich Information Center for Protein Sequences

The data from the Munich Information Center for Protein Sequences database yielded some iteresting information about YFL030W. This website tells us that the protein product is catergorized as an aminotransferase with a class-V pyridoxal-phosphate attachment site. The functional categories it was related to were amino acid biosynthesis, amino acid degradation, and nitrogen and sulfur utilization. This relates back to to the DIP search, as it is clear these searches find some relation between the gene product of this annotated gene and phosphate kinases, due, presumably, to the site on the protein. The association of YFL030W to the above functional categories was not previously recorded.

PDF Files on Protein Connections

Searches for YFL030W from the following files yielded no matches: Aging (PDF format), Membrane (PDF format), and Degradation (PDF format).

YRC Two-Hybrid Analysis

The University of Washington's Yeast Resource Center's two-hybrid analysis yielded no hits of YFL030W.

WIT

There was no information of use relating to YFL030W in the What is There? website.

Experiments/Conclusions

The gene product of YFL030W was previously predicted to be involved in aerobic cellular respiration. This information, however, indicates that it is an aminotransferase. Based on the searches above, it is a safe assumption that the DIP searches may not be completely accurate. The first experiment I would perform on this protein would be a test of this presumed presence of a "pyridoxal phosphate cofactor". I would clone the gene then test this interaction by any number of different ways, including Western blotting or immunofluorescence. Confirmation of this need for a cofactor would certainly be an important step in determining the function of protein YFL030W. If this interaction turned out to be real, further investigations would be needed to determine the interactions of this protein before and after the cofactor is added. Following this, understanding the localization of this protein and possibly further examining its relationship between PHO85 and DUN1. Immunofluorescence of the cell dealing with both of these proteins could reveal these interactions.

Because the TRIPLES database hinted that this may be a protein essential for cell viability, knocking out this ORF and testing for cell viability and general cell characteristics (i.e. reproduction) would be useful. Immunoprecipitation and yeast two-hybrid (Y2H) studies with this gene would be interesting, though other than the PHO4 transcription factor found in the immediate connection to YFL030W, no other information dealing with transcription factors was found. Using PHO85 and DUN1 as bait proteins would be helpful in determining whether there is an interaction between these two genes and our unannotated one. Experiments that would also be useful go back to the gene expression work. These experiments indicate the involvement of this protein in aerobic respiration, and, although no searches above confirmed this prediction, these data could still prove to be useful. By growing yeast cells in aerobic and anaerobic conditions, as well as with ethanol or acetate added and performing the above experiments, some of the interactions can be solidified.

Although not much is known about the function of YFL030W, deductions from its structure and interactions with other proteins create a patchwork. Using the experiments discussed above, the actual function of this protein could be deduced.

References (Websites Used):

DIP

Yale Gerstein Lab

TRIPLES

Munich Information Center for Protein Sequences

two-hybrid analysis

What is There?

Saccharomyces Genome Database

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