Figure 1. This screen shot shows no protein interactions between Cyt1 and another protein. Since proteins sometimes bind one way, but not the other (i.e. RPC19 binds to bait RPC40 but RPC40 does not bind to prey RPC19), I checked to see if Cyt1 interactions were detected when Cyt1 was listed as prey, instead of bait. There were no protein interactions when CYT1 was used as the activation-domain-bound prey.

This image makes interactions between the bait Cyt1 and the ORFs of other yeast genes in the Y2H method appear absent, assuming a genomic library was transformed into plasmids where CYT1 was being used as bait, CYT1 does not appear to interact with any other yeast proteins. Since the interactions detected using this method must occur inside the nucleus of the yeast cell, and CYT1 is located in the mitochondria, it is possible that CYT1 does not work well inside the nucleus. A conformational change in structure could cause a misshapen protein which, inside the nucleus, does not bind to the proteins with which we might expect CYT1 to interact. CYT1 as a "prey" protein might also be misshapen and thus behave differently inside the nucleus than in the mitochondria. Another possibility for the lack of interaction is that the Y2H experiment has yet to be run with Cyt1.

Figure 9. The evidence that other yeast proteins are involved in the same biological process of electron transport, molecular function of oxdative phosphorylation and the same cellular component of inner mitochondrial membrane is well supported. There are over 1000 proteins involved in the cellular component of the mitochondria and over 100 of these are localized to the inner mitochondrial membrane (see right). There are also many other proteins involved in the same biological processes as Cyt1 (see left).

In the 42 gene products associated with yeast cytochromes and the 55 gene products associated with yeast mitochondria (SGD, 2004), it is likely that many interactions with CYT1 protein occur. The data found from the DIP database do not describe the entire story of CYT1 protein interaction. There are at least two possible explanations for the fact that no data is exhibited in the interaction illustration of Cyt1 in the DIP database. Databases are being further developed constantly. DIP may not yet have tested the interactions between Cyt1 and all other yeast proteins. Another possibility is that Cyt1 has very limited interaction with other proteins, since Cyt1 serves as an electron transport molecule in the cytochrome c reductase complex. This could potentially mean that Cyt1 only interacts with one molecule which it transports from ubiquinol to cytochrome c.

No results were found in the PDFs for membranes, degradation or aging proteins. I would have expected Cyt1 to appear in the membrane protein PDF, although perhaps inner mitochondrial membrane is structurally more similar to a membrane bound protein, than a membrane protein, in this case.

Y2H protein interaction has probably not yet been investigated as there appear to be no interactions in this image and we can be fairly certain that Cyt1 interacts with at least one other protein in order to properly carry out its function. It is important to remember that Cyt1 is a mitochondrial protein and so it may not function, or interact, fully in the nucleus, which is where the Y2H method takes place.

It is fairly clear, based on data from the SGD site, that this protein is involved in oxidative phosphorylation and located in the mitochondria of yeast. Investigations in proteomics show that there is much more to be figured out about the function of even relatively well understood proteins.

Because the YOR066W protein hybridizes with the YLR297W protein we now have a new ORF to investigate.

Interestingly, YOR066W does bind to CDC28 as a prey protein. There are two other proteins which are now of interest. CLB2 and CLB5 also bind to YOR066W in the Y2H experiment. There are interactions between YOR066W and known protein products. At this point we have three strong leads as to a potential function of YOR66W in its binding to Cdc28, Clb2 and Clb5.

Given that there is no information regarding the direct interaction of YOR066W and other proteins, from the DIP database, it is useful to see interactions of known proteins with a non-annotated gene protein products. It is important to note that the design of this graph does not necessarily mean that there is no interaction between YOR066W and Cdc28. The red edge from Clb5 to YOR066W signifies that the interaction is from an unverrified, high throughput method. The green line between Clb5 and Cdc28 is better established and verified with computational methods. The data do not eliminate the possibility of more interaction of the YOR066W protein.

Experiment 1: YOR066W, as seen in the Y2H method, bound to protein kinase regulators, in the case of Clb2 and Clb5. In the Y2H method, YOR066W bound to a kinase, which phosphorylates amino acids, in the case of Cdc28. Mike Snyder's kinase functionality experiment would be a way to test the function of YOR066W. First, it would be necessary to make microwells to which different substrates would be covalently linked. One substrate would occupy each well. The YOR066W protein would then be added to each well along with radioactive ATP. After incubation, the microwell arrays would be imaged with a phosphoimager to see where radioactive Phosphates where incorporated into the substrates in the silicon. If YOR066W binds to kinases, this would suggest a function as a kinase substrate in the yeast genome.

Experiment 2: Another possibile method to determine proteomic information about YOR066W is the isotope-coded affinity tags (ICAT) method. Since we are working with yeast, this experiment is not limited by the conditions of growing cells in heavy isotope. First, it is necessary to pick two conditions that will form different populations of cells. We could incubate one set of yeast cells in normal growth conditions and use the heavy ICAT labeling reagents to label these cells. Another set of yeast cells could be grown in a chemical condition that arrests the cell cycle at a particular phase. The cysteines of this second group of cells would be labeled with the light ICAT labeling reagent. After being labeled, the two cell populations would be mixed and biotin/avidin-purified. The labeled peptides would be put into a mass spectrometer to develop heavy to light protein ratio. Provided the protein ratios are big enough for the mass spec. to detect, we will see whether the cells that were arrested in a specific cell cycle produced fewer copies of any proteins. Of particular interest would be the affects of an arrested cell cycle on the YOR066W protein for which ratios could be compared to see if YOR066W plays a role in cell cycle.