This web page was produced as an assignment for an undergradute course at Davidson College
(Please read a general description of Yal011w, which can be found as a link from my
homepage, if you are not familiar with this ORF)
Protein-protein interactions can be very useful in identifying possible functions of unknown proteins such as Yal011w. Although neither Eisenburg's database <http://dip.doe-mbi.ucla.edu/dip/Search_DIP.cgi> nor Stanfield's database <http://depts.washington.edu/%7Eyeastrc/th_11.htm> lists proteins that interact with Yal011w, the Yeast Proteome Database <http://www.proteome.com/databases/YPD/YPDsearch-quick.html> lists six proteins that interact with Yal011w. The six proteins that are known to interact with Yal011w come from a study done by Ito et al. (2001) in which a massive two-hybrid screen was done on all 6000 proteins in the yeast proteome in every possible combination! That is a lot of work. Their results showed that Yal011w interacts with Ser3p, Inh1p, Kel1p, Pac1p, Rga1p and Yap5p. Although these results are impressive, a closer look at these data shows the interactions must be taken with caution.
Yal011w is a mitochondrial protein whose sequence is consistent with a transmembrane protein. As such, any protein not localized to the mitochondria that interacts with Yal011w in a yeast-two hybrid screen must be treated as suspect. Kel1p and Rga10, for example, are two proteins involved in polarizing the cell during mating through their interations with the cytoskeleton. The localization of these proteins, however, is in the cytosol and it is therefore unlikely that Yal011w interacts with these proteins under normal cellular conditions. The interaction between Yal011w and Yap5p under normal cellular conditions is also questionable as Yap5p functions in the nucleus as a transcription factor. Because spatial localization would not support the tenet that Yal011w interacts with these proteins, one could only verify these interactions with a functional test. In situ fluorescent hybridization with antibodies against Yal011w and its supposed ligand would support their interaction if both antibodies localized to the mitochondria under normal conditions. Varying the color of the fluorescence for each antibody would give a standard color pattern to expect if these proteins did indeed interact.
A much more likely interaction that can be gathered from the results of Ito's study is the interaction between Yal011w and Ser3p. Ser3p has an unknown location but its function highly suggests that it localizes with Yal011w to the mitochondria. This protein convert intermediates of energy metabolism into serine. Although the intermediates that Ser3p converts to serine (3-phosphoglycerate and 3-phosphopyruvate) are intermediates from glycolysis, which occurs in the cytoplasm, there are at least two enzymes in glycolysis that reside on the mitochondrial membrane, name pyruvate kinase and hexokinase.
The interaction between Yal011w and Inh1p is also highly plausible. Inh1 lcalizes to the inner mitochondrial membrane and is an inhibitor of ATPase activity. That is, it functions to block the synthesis of ATP, possibly during times in between meals when ATP is not being made but rather being incorporated into glycogen.
Future studies that would be beneficial to further characterize Yal011w would most profitably be based on testing interactions between Yal011w and proteins that localize to the mitochondria. The interaction between Yal011w and Ser3p is particularly interesting because it shows that Yal011w interacts with a protein involved in metabolism. Proteins in metabolism, moreover, are highly regulated so that the correct metabolic pathway is turned on at the proper time. Hence Yal011w may bind with Ser3p to induce a conformational change. A good way to test this possibility would be to crystallize Ser3p in isolation and in the presence of Yal011w. If the crystallized structure of Ser3p differs in the presence of Yal011w, it is plausible that Yal011w, like other mitochondrial proteins, regulates a metabolic pathway through inducing a conformational change in one of the participating enzymes. Studies should also be done to see what kinds of modifications Yal011w undergoes. In the figure below, Yal011w does not migrate according to its proposed pI (see red square). Thus, Yal011w may be a phosphorylated protein. To test whether Yal011w undergoes phosphoylation, one could attach the protein to a the polymerized chip PDMS, as Mike Snyder has done, and incubate the protein will the 122 known kinases in yeast. If radioactive ATP was used as a substrate, radioactive Pi would be taken up by Yal011w and would be bound to the chip, which could then be assessed for radioactivity. If Yal011w is phosphorylated by one of the kinases, then radioactivity would be detected on the chip after all substrates had been washed off the chip.
This figure was taken from <http://expasy.cbr.nrc.ca/cgi-bin/ch2d-search-de> and permission is being sought to keep it here
1) Yeast Proteome Database [online database]. Available from: <http://www.proteome.com/databases/YPD/YPDsearch-quick.htm
2) Ito et al. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A. 98(8):4277-8
Click here to return to the Davidson College webpage at bio.davidson.edu/genomics/