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My favorite yeast proteins
My search for information on Kex2 began at Mike Snyder’s TRIPLES database. Unfortunately, this search was fruitless. I then searched the PDB in hopes of finding a CHIME image of the protein. This was also fruitless. Stan Field’s yeast two-hybrid data did not use Kex2 as bait, nor was it ever identified as prey. My first positive result came searching the DIP database. The database returned an interaction map shown below.
Kex2 is the red node, and is shown to interact with one protein, identified as BFA1, though it should be noted they use a thin line, indicating a low probability of actual interaction. A PubMed search returned abstracts identifying this protein as being involved in cell cycle regulation(abstract here). This may suggest that Kex2 has some additional role in cell cycle regulation. The Curagen database indicates that Kex2 does not interact with any other proteins(here). We know this is not true, but the database is based on limited data. I then hoped to identify Kex2 in a 2d gel database. In the PROWL database, I found the MW of Kex2 to be 90000 and the pI to be 4.8(here). Unfortunately, no spots were identified at the corresponding points(gel shown here). There really isn’t much information available about Kex2 in these databases. To find out more about the Kex2 protein, I would start by performing a yeast two-hybrid experiment using Kex2 as a bait protein to find out exactly which proteins interact with it. We would expect to find Kex2 interacting with the proteins it cleaves, but we would also be looking to find proteins that may modify Kex2, or act as coenzymes. I would then perform experiments using the ICAT method, observing relative protein levels under varying conditions. In particular, I would want to verify the DIP data suggesting Kex2 has a role in the cell cycle by observing relative Kex2 at different points in the cell cycle. I would also want to use the ICAT method to determine if Kex2 is post-translationally modified in any way, such as phosphorylation. Under the current model of µ-factor maturation, we would not expect to see any modification of Kex2. We would expect it to be active at all times, its behavior being modified only by the presence or absence of its substrate, the µ-factor.
SCYNL234W is a somewhat more interesting story. Once again, TRIPLES and PDB returned nothing. The yeast two-hybrid system returned three hits however. All three are in the recent data link from the main Y2H page. The first YNL234W hit is as a prey ORF for CHK1 bait. A PubMed search identified CHK1 as a kinase involved in the cell cycle arrest response to DNA damage(here, and here). The second hit is also as a prey ORF to the bait protein CKB1. A PubMed identified CKB1 as the regulatory subunit of caesin kinase 2(CKII), which has a few different functions. Interestingly, one of those functions is cell cycle control, specifically in exiting G1 and G2(here). The third hit is as a prey ORF for the bait protein YPT31. PubMed revealed YPT31 as a golgi protein involved in vesicular transport(here). It should be noted that two of the three hits are involved in cell cycle regulation. This may indicate a possible role of YNL234W in cell cycle signaling. Interestingly, the expression data did not support this, though the limitations of those data have been noted. DIP identified YNL234W as interacting with four different proteins, each involved in an intricate signaling pathway(click on graph button in upper right of this link).
Three of the four identified interactions(KC2B, YIV8, and IMB3) are shown with the thin bar, indicating a low probability of interaction. One of them is shown with the next thinnest bar. This protein is CHK1, which Y2H also indicated. I then went to search Curagen, which indicated that YNL234W did not interact with any other proteins(here). The PROWL database had not identified the MW and pI of YNL234W, making it unidentifiable on their 2D gels. The cumulative data found in these sites seem to indicate through a number of independent experiments that YNL234W may play a role in cell cycle regulation. This is quite interesting since none of our expression data or sequence analysis posed this as a possibility, though they do not rule it out. One reason the expression data may not suggest this is that expression levels may not be the primary activity of the protein. The proteins it appears to interact with are kinases, leading us to conclude that the primary regulator of this protein may be its phosphorylation state.
To find out more, I would start by performing Y2H experiments with YNL234W as the bait protein, in hopes of confirming previous data. I would then perform ICAT experiments examining protein levels, and phosphorylation states at different points in the cell cycle, and also in response to radiation damage to DNA. If YNL234W is indeed involved in the cell cycle, as it appears to be, the most critical data will probably be its phosphorylation state under these conditions, given that the proteins with which it appears to interact are kinases.
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Many thanks to Dr. A. Malcolm Campbell for his guidance in this endeavor as well as others.