Proteomics of LAS17 and YOR186

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

Thus far in my studies of genomics, I have examined the DNA sequence and protein functions of one annotated and one unannotated gene in the baker's yeast (Saccharomyces cerevisae) genome.

Las17 is a gene that encodes a protein that assists with actin formation. In this website, I will examine the protein characteristics and interactions of Las17.

Yor186 is an unannotated gene that has no known function. In previous websites, I have used genomic techniques to predict the function of this gene. In this website, I will further examine my hypothesis using proteomics and suggest an experimental design to understand the function of Yor186.

Protein Data Bank

Figure 1. Schematic of the the BBC1 component of the Sh3 domain of S. cerevisae from PDB. The pink peptide chain of proline originates from LAS17.

Although PDB did not have any information about the LAS17 protein specifically, recent additions to the Sh3 domain indicate that a proline peptide chain is involved with the formation of this domain.


The Sh3 domain of the yeast genome acts by selectively integrating signals from various transduction pathways to physically assist protein interactions or protein interaction with their target molecule. (SGD)


Other general protein information for LAS17 is that it has a molecular weight of 67,571 Da and an isoelectric point of 10.05. (SGD)


TRIPLES Results for LAS17

Figure 2. Data from TRIPLES that shows where the transposon mTn was inserted and how the subsequent disruption of the gene affected the cell and its phenotype. These results yield insights into subcellular locations and where the proteins are expressed.

To learn more about the position, location, and function of LAS17 with in the S. cerevisae cell, I examined the information about subcellular localization of LAS17. These two different results are a consequence of mTn inserting in different locations of the gene. From this information, I have determined that LAS17 appears to function in the cytoplasm during all cellular stages. Results from V177A4 suggest that perhaps LAS17 is more active in the cells during budding, cellular replication.
Database of Interacting Proteins
Figure 3. Database of Interacting Proteins results for LAS17.
This initial figure produced by DIP immediatly shows that Las17, the red node in the center of Figure 1, is involved in many different protein interactions with other proteins that are involved with actin production. We would expect these results because as shown in my webpages about the gene and the protein, the Las17p protein is a regulator protein that will interact with many other proteins to form the correct phenotype of actin production.
Interacting First Shell Nodes as Determined by the Database of Interacting Proteins
Table 1. Proteins that interact with LAS17 during actin filament formation. Information obtained from SGD.


Protein Function


Protein binding and bridging
Rgd1p Involved with actin formation and organization
Pep1p Signal sequence binding
Nsg1p Protein binding


Actin filament organization
YHM2p DNA binding
Rpn12p Endopeptidase activity
Cap2p Actin filament binding
Ysn1p Unknown
Lin1p Protein binding
Aac3p ATP:ADP antiporter activity
Sho1p Osmosensor activity
Hxt6p Fructose, glucose, mannose transporter
Gal2p Galactose and glucose transporter activity
Sqt1p Unknown
Vps5p Contributes to protein transporter activity
Chc1p Structural molecule activity
Hxt7p Fructose, glucose, mannose transporter
Ent1p Actin cortical patch assembly
Lsb5p Actin filament organization
Bbc1p Myosin 1 binding
Vma6p Hydrogen-transporting ATPase activity
End3p Actin cytoskeleton organization, cell wall morphogenesis


Protein Function
Hof1p Cytoskeleton protein binding
Arp3p Actin binding
Bzz1p Actin filament organization
Sna3p Unknown
Myo3p Microfilament motor activity
Myo5p Microfilament motor activity
Sla1p Cytoskeleton protein binding
Rbd2 Unknown
Rvs161p Cytoskeleton protein binding
Ysc84p Actin filament organization
Lsb3p Actin filament organization
Vrp1p Actin binding
Rvs167p Cytoskeleton protein binding
Pho84p Inorganic phosphate transporter activity
Sla2p Transmembrane actin binding
Sac6p Actin filament binding
Pin3p Unknown
Act1p Structural constituent of cytoskeleton
Lsb1p Unknown
Cof1p Protein binding
Rsp5p Ubiquitin protein ligase activity
Myo4p Microfilament motor activity
Bem1p Protein binding


As expected, many of these proteins are involved with actin synthesis and organization as well as assisting with protein binding. This is an example of the complexity of protein interactions within the cell for a seemingly simple process of creating a new actin filament.
Y2H Results for LAS17

Figure 4. Y2H results for LAS17 using this protein as a bait and showing other proteins that LAS17 bound to as prey.

Y2H results show that LAS17 works as a bait protein in this method for five different prey proteins. These results demonstrate the overlap of Y2H and DIP, but also the need to use both databases when determining protein interaction. DIP results showed many more proteins that are predicted to interact with LAS17, but did not include NSG1. Therefore, DIP does not necessarily reflect all proteins that interact with LAS17. Furthermore, PRP40 is not included in the DIP results as we would expect from Y2H results, but we may be able attribute this to the fact that this interaction was only determined when PRP40 was used as bait and not vice versa.
Other Databases Searched
Database Results
ExPasy No new information.
KEGG No data.
SGD No new information.
Protein Information Resource No new information.
ProFound/PROWL No data.
Facts Obtained From Protein Information Resource
  • YOR186w has a predicted transmembrane domain from amino acids 94 -110.
  • YOR186w is a single-pass membrane protein as designated by its sub-cellular location.
  • YOR186w molecular weight is 16180 Da.
  • YOR186w has an isoelectric point at 4.7.
These results obtained from the Protein Information Resource are surprising and new. On October 31st, new data was added that suggested that YOR186w may have this transmembrane domain. These results are contrary to my results using DNA analysis, more specifically, the Kyte-Dolittle Graph.
ExPasy 2-D Gel

Figure 5. ExPasy results for YOR186w. It appears that in the black box that surrounds the area where YOR186w should be located has several small black dots indicating that low levels of protein were found there. YOR186w may or may not be represented on this gel because it is difficult to distinguish.

TRIPLES Results for YOR186w

Figure 6. TRIPLES results for YOR186w that shows where the transposon mTn was inserted and how the subsequent disruption of the gene affected the cell and its phenotype. These results yield insights into subcellular locations and where the proteins are expressed.

Of the few TRIPLES results available for YOR186w, I was surprised to find protein information that suggested that this protein was found within the cytoplasm during budding and the vegetative state. The transposon inserted fairly early into the ORF at codon number 6, which allowed the detection of LacZ expression. These results are contrary to other protein information available and described on this webpage, but it is possible that TRIPLES was only able to assay the cytoplasmic side of YOR186w if it is in fact a transmembrane protein.
Y2H Results for YOR186w

Figure 7. Y2H results for YOR186w.

These results indicate that Y2H assays yielded one protein interaction that involved YOR186w. As a bait protein, YOR186w bound to YDR132c. This protein is another hypothetical protein similar to YOR186w with unknown molecular function or biological process. Researchers are aware that YDR132c is present in the cytoplasm and in the nucleus. (Yeast Resouce Center)
DIP Results for YOR186w

Figure 8. Database of Interacting Proteins results for YOR186w.

DIP results indicate that YOR186w likely interacts with several other proteins since they do have it available as a node, but they do not yet have any information about protein interactions even within the 1st shell nodes.
Other Databases Searched
Database Results
KEGG No data.
SGD No interaction data.
Protein Data Bank No data.
ProFound/PROWL No data.
Conclusions of the Function of YOR186w
My proteomic results are surprising. During the analysis of the gene and DNA sequence, I used a Kyte-Dolittle Graph to predict if YOR186w had any transmembrane regions and determined that the sequence did not. However, during my proteomic research on this ORF, I found that it likely had transmembrane properties. Unfortunately, the proteomic databases available online had little more to offer about this ORF, although, we still may be able to make some conclusions about YOR186w. This ORF appears to have some transmembrane property that combined with information regarding its expression profile may provide enough background to make some predictions about its function. Since this protein is predicted to be a transmembrane protein and appears to be expressed similarly to other genes known to be involved with transcription and translation, YOR186w may be a nuclear membrane protein that assists or regulates mRNA or DNA replication protein movement through the nuclear membrane. This explanation would also explain why TRIPLES may have assigned the protein to the cytoplasm of the cell.
Possible Experiment to Determine the Function of YOR186w in Saccharomyces cerevisae

My first step would be to specifically determine the cellular location of YOR186. I would try to use first immunofluorescence provided that a suitable antibody for YOR186w exists or could be developed. Next, the antibody would need to have an epitope tag that was labeled with an immunofluorescent that would glow wherever YOR186w was found within the cell.

My next step would be to determine more proteins that interact with YOR186w. Techniques I could use include immunoprecipitation, crystallization, or the Y2H method. We could use immunoprecipitation to precipitate proteins that physically interact or bind with YOR186w. This requires that an antibody binds with YOR186w that when centrifuged would pull this protein and any physically bound proteins so that they may be separated out of solution. We could then identify these proteins by sequence analysis. Crystallization of YOR186w could be used to determine what molecules interact with the protein active site. We would crystallize YOR186w and then in solution add many different molecules or other proteins that fit within the active site of the protein. The final method I would recommend for identifying proteins that interact with YOR186w is the Y2H method. From the results I have found using this method, it does not appear as if YOR186w was ever used as the bait protein. Using this method, we may be able to determine which proteins interact with YOR186w.

Once we have determined a location and possible protein interactions, we may be able to final deduce its function when used in conjunction with data about its DNA sequence and expression profile. Thus far, little information appears to exist about YOR186w. This is why I would encourage the use of general methods to learn more about the protein. From my predictions, I would assume that we would find that YOR186w is a nuclear membrane protein. I would also expect to find that this protein interacts with other proteins involved with DNA transcription and subsequent translation of mRNA.

One method to use that would refine the prediction of YOR186w's transmembrane region, would be to use a method developed by Michel Desjardins's group at the University of Montreal. First, we would need to develop a method to separate the nuclear membrane from the cell, but if this could be accomplished, we would have data that may or may not support this prediction. We could subject the membrane to Triton X-114, a detergent that cleaves proteins at the membrane. If YOR186w was isolated from this sample, we could reasonably assume that this protein is at least bound to the membrane. We could run subsequent analyses using proteases to determine if the protein had components on either side of the membrane by cleaving proteins outside of the membrane before isolating the membrane and running the same test. If part of YOR186w is present in the results, we may assume that it is a transmembrane protein.

Databases Used

Database of Interacting Proteins - Accessed 11/16/2006

ExPasy - Accessed 11/16/2006

KEGG - Accessed 11/16/2006
Protein Data Bank - Accessed 11/16/2006
Protein Information Resource - Accessed 11/16/2006
PROWL/ProFound - Accessed 11/16/2006
Saccharomyces Genome Database - Accessed 11/16/2006
TRIPLES - Accessed 11/16/2006
Y2H - Accessed 11/16/2006
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© Copyright 2006 Department of Biology, Davidson College, Davidson, NC 28035