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


My Favorite Yeast Proteins: Exploring the Roles of CDC28p and YBR161Wp

CDC28p: Gene Products CKS1, FUS1/SGV1, and FAR1.

Triples Database.

I found no information for any of CDC28's gene products (Triples database, 2001; http://ygac.med.yale.edu/triples/basic_search.asp).

Database of Interacting Proteins (DIP).

I searched this database for cdc28 and found several hits for each of its protein products.  I focused on the cell division control protein CKS1.  Here are the 9 proteins it interacts with:



CENTERED ON
Links Interaction
information
PIR SWISSPROT GENBANK NAME
- OKBYS1 CKS1_YEAST 116484 cell division control protein CKS1
INTERACTS WITH 9 PROTEINS
S14165 CG21_YEAST 82956 cyclin B1
S14166 CG22_YEAST 82957 cyclin B2
A60048 CG23_YEAST 419827 cyclin B3
S45591 RS8_YEAST 603340 ribosomal protein S8.e, cytosolic
S53479 YAJ7_YEAST 1078469 FUN55 protein
TVBY8 CC28_YEAST 66745 protein kinase cdc28
S38189 DUT_YEAST 487080 dUTP pyrophosphatase precursor, mitochondrial
S48765 YD78_YEAST 629935 probable membrane protein YDR078c
B60048 CG24_YEAST 419828 cyclin B4

Figure 1. Table of proteins CKS1 interacts with. More figures of CDC28 interactions can be found at this link: http://dayhoff.mbi.ucla.edu/~jdip/img/.  With permission from Ioannis Xenarios (DIP, 2001; http://dip.doe-mbi.ucla.edu/).


 



These findings were consistent with the preliminary searches from my previous web pages.  (DIP, 2001; http://dip.doe-mbi.ucla.edu/).

YRC Two-Hybrid Analysis.

The Y2H method confirms that CKS1 (prey) interacts with CLB1, CLB2 and CLB3 (baits) (YRC, 2000; http://depts.washington.edu/%7Eyeastrc/th_11.htm). The full paper can be seen here: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10688190&dopt=Abstract.

Schwikowski PDF Files.
In the PDF files of protein-protein interactions, CDC28 was found to be a central player in a large and complex circuitry.  My protein interacted with more than 10 different proteins; most of these interactions were between proteins with identical cellular roles but different localizations (lime green lines) (Schwikowski et al., 2000; http://www.bio.davidson.edu/courses/genomics/Benno/NB_Figure1color.pdf).  Also, an interesting discrepancy arose here: on these complex maps, CDC28 is shown to interact with CKS1, where previous documentation indicated that CKS1 is a functional subunit of CDC28 (SGD, 2001; http://genome-www4.stanford.edu/cgi-bin/SGD/locus.pl?locus=CKS1). This finding reiterates that this map should be used as a starting place for further exploration, not as a source of absolute fact.
CDC28 was also found to interact with similar proteins in each of the other PDF files:
http://www.bio.davidson.edu/courses/genomics/Benno/membrane.pdf
http://www.bio.davidson.edu/courses/genomics/Benno/aging.pdf
http://www.bio.davidson.edu/courses/genomics/Benno/degradation.pdf
This confirms the ubiquity of CDC28 and also its widespread impact.  The full paper can be seen here: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11101803&dopt=Abstract.

Protein Database.
I found this Chime image of crystallized CKS1:


Figure 2.  Chime file of CKS1, the protein kinase subunit of CDC28.  Note the three distinct pieces, as well as a long side chain.  Though this isn't the complete protein, it is substantial in size, suggesting it can serve several different functions under different conditions (PDB, 2000; http://www.rcsb.org/pdb/cgi/explore.cgi?pid=144601005234799&pdbId=1QB3).

GenTHREADER.

Using the PSIPRED V2.1 Server, I found the following graphical views ofCDC28:
http://bioinf.cs.ucl.ac.uk/cgi-bin/psipred/graphics/nph-view.cgi?id=10052787589595.  Though I wasn't able to make much sense of this information, I found it impressive that the model generated these outputs based solely on amino acid sequence (PSIPRED V2.1 Server, 2001; http://insulin.brunel.ac.uk/psipred/).

Snyder: Kinase Functionality.

In a recent paper, a group of researchers implemented a high throughput method to survey the function of kinases.  As my gene is a cyclin-dependent kinase, I searched their data, but they found CDC28 to be inactive in their assays.  The authors hypothesized that this protein must have been lacking its activating cyclins (Zhu et al., 2000; http://www.nature.com/cgi-taf/DynaPage.taf?file=/ng/journal/v26/n3/full/ng1100_283.html).

WIT Database.

I found no information for any of CDC28's gene products here (http://wit.mcs.anl.gov/WIT2/CGI/index.cgi).

Enzymes and Metabolic Pathways Database.

The EMP database yielded no information for any of CDC28's gene products. (EMP, 2001; http://emp.mcs.anl.gov/cgi-bin/map_search.pl).

Conclusions.
CDC28 is a well-documented gene, so I found little new information about its protein products.  However, it became clear that CDC28 is an essential protein to normal cellular processes; its impact is so far-reaching, slight variations is either its functionality or its concentration would likely have disastrous results.  We already know that a deletion of CDC28 yields a yeast cell inviable, but another interesting experiment might test how different concentrations of CDC28 impact the cell.  We have also already seen from microarray data that only slight variations in CDC28 have huge influences on other proteins in the cell.  What might happen if a cell was subjected to a huge influx of CDC28? Would the cell be overwhelmed with signal it would shut down and cease mitosis altogether? Or, would the cell (and its daughter cells) just mitotically divide like crazy and only lose signal after an extended period of time?  An experiment of this sort might give us some appreciation of the complexity and interconnectedness of the cell web by demonstrating how interdependent each protein is on each other protein.


YBR161Wp:

From my first 2 web assignments, I suspected my non-annotated gene to be involved in DNA replication.  Preliminary searching of SGD indicated that YBR161W protein is similar to SUR1, a gene product involved in maintenance of phospholipid levels (SGD, 2001; http://genome-www4.stanford.edu/cgi-bin/SGD/locus.pl?locus=SUR1).  For each database, I searched both YBR161W and SUR1.

Triples Database.

I found no information for YBR161W, but SUR1 was disrupted by the mTn method.

TN7-62G7:     Potential ORFs Disrupted by mTn Insertion


Gene or ORF (plus synonyms) mTn insertion/ORF features Chr. Chr. Insertion Coord. Insertion point (codon #) Total ORF length (amino acids) Repeated Gene LacZ Orientation
SUR1/BCL21/CSG1/LPE15/YPL057C In frame XVI 452423 211 382 may be sense

Gene Expression Data


Growth condition LacZ expression level Background strain
vegetative faint blue Y800
sporulation faint blue Y800

Figure 3. Effects of mTn transposon insertion on Sur1 gene.   (Triples Database, 2001; http://ygac.med.yale.edu/triples/get_clone_info.asp?cloneid=TN7-62G7).

While this was quite interesting, it yielded no useful information about my non-annotated gene.

Database for Interacting Proteins (DIP).

Searching DIP for YBR161W yielded the following protein interaction:



CENTERED ON
Links Interaction
information
PIR SWISSPROT GENBANK NAME
- S46032 YB11_YEAST 626229 SUR1 protein homolog YBR161w
INTERACTS WITH 1 PROTEINS
S57112 JSN1_YEAST 1077899 JSN1 protein

Figure 4. Interactions for YBR161W. Permission pending. (DIP, 2001; http://dip.doe-mbi.ucla.edu/).

Further DIP searching revealed JSN1 is a RNA-associated protein (DIP, 2001; http://dip.doe-mbi.ucla.edu/).  A study shows a mutant strain overexpressing JSN1 was more sensitive to benomyl, a microtubule-destabilizing drug.  This suggests JSN1 plays a role in anaphase, namely spindle elongation (Machin et al., 1995; http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&uid=8534919&dopt=r). These findings suggest that YBR161W may also play a more specific role in the mitotic stage of anaphase.

YRC Two-Hybrid Analysis.

The Y2H method database revealed no interactions for either YBR161W or SUR1.

Schwikowski PDF Files.
I found no documentation of protein-protein interactions in the Schwikowski PDF files (Schwikowski et al., 2000; http://www.bio.davidson.edu/courses/genomics/Benno/NB_Figure1color.pdf; http://www.bio.davidson.edu/courses/genomics/Benno/aging.pdf; http://www.bio.davidson.edu/courses/genomics/Benno/membrane.pdf;
http://www.bio.davidson.edu/courses/genomics/Benno/degradation.pdf).

WIT Database.

I found no information for either YBR161W or Sur1 here (http://wit.mcs.anl.gov/WIT2/CGI/index.cgi).

Enzymes and Metabolic Pathways Database.

The EMP database yielded no information for either Sur1 or YBR161W. (EMP, 2001; http://emp.mcs.anl.gov/cgi-bin/map_search.pl).

GenTHREADER.

Using the PSIPRED V2.1 Server, I found the following graphical views of YBR161W:
http://bioinf.cs.ucl.ac.uk/cgi-bin/psipred/graphics/nph-view.cgi?id=10052787319587  (PSIPRED V2.1 Server, 2001; http://insulin.brunel.ac.uk/psipred/).

Experiments.
    The best lead I found concerning my non-annotated gene is its documented interaction with JSN1, a protein that may be both an RNA-binding protein and involved in spindle elongation during anaphase.  If JSN1 is able to play multiple cellular roles, it becomes unclear which process YBR161W is interacting with.  I would first need to answer the question, where is YBR161W interacting with JSN1?  Using a method similar to the bar code analysis only on a smaller scale, I could see what cellular process(es) YBR161W is involved in.  If I found that YBR161W is involved in cell division by clustering with other genes that performed similar functions, I could design further experiments to see if my protein participates in anaphase.   I would first need to design an experimental condition that induces mitosis.  If alpha-factor induces meiosis, then there may exist a complementary pheromone to induce mitotic division.  I would then create a knock-out mutant missing my ORF and complementary protein.  If these cells were unable to undergo mitosis, I would know that YBR161Wp was an essential protein and hence must be somehow involved in the process of cellular division.  I would also look for any aberrations in cell division, i.e. if cells divided particularly slowly, if aneuploidy was a significant result, etc.  If none of these results were apparent, I wouldn't be able to make any positive conclusions concerning YBR161W's role in mitosis, only that I needed to investigate further.
    I could also design a monoclonal antibody that only bound to my protein.  Then, I could find some screen to look for my antibody, which could ideally give me a quantitative measure of how much of my protein was present in cells, especially during mitosis.
    If I did find a positive indication that YBR161Wp plays a role in mitosis, I would attempt to qualify what that role was.  I could fluorescently label my protein/antibody and induce mitosis in these yeast cells.  Then, I could kill the cells at different stages of the mitotic process.  Looking under a microscope, I would ideally be able to discern where my protein was interacting, i.e. in which stage of mitosis.  If I was to say with some certainty that YBR161Wp participates in anaphase, I'm not sure how any proteomic methods could help me say what exactly it was doing.
    If none of this worked, I would have to go back to the possibility that YBR161Wp was interacting with JSN1 as an RNA binding protein, not a protein involved in spindle elongation.  With the same idealized methods from above, and also labeling protein and fixing cells at different stages of mitosis, I could potentially discover where the protein was isolated within the cell, i.e. nucleus, ER, cell membrane.  This could lead to a more definitive identification of YBR161's cellular role.
    The problem with most of the methods we studied in the Proteomics chapter is that they each take  a large population of proteins and try to make generalized statements about the entire proteome; here, I would like to say something about a single protein during a single cellular process, both qualitatively and quantitatively.  I'm not sure if any of my described methods would be physically possible, but at least in an idealized situation, these are the questions I would like to answer.
 

Conclusions.
Proteomic searches have shed more light onto YBR161W's role in cellular processes.  Interestingly, its sequence similarity to SUR1 led to several dead-ends in my online searches.  I originally picked this ORF because of its close proximity to CDC28; after lengthy exploration, it turns out this non-annotated protein may play a role in mitosis, much like CDC28 after all.  When comparing the two, it is obvious that CDC28 is a larger blanket protein, while YBR161W does not.



References.

1. Triples Database. 2001. <http://ygac.med.yale.edu/triples/basic_search.asp>  Accessed 6 Nov 2001.

2. Database of Interacting Proteins.  2001. <http://dip.doe-mbi.ucla.edu/>  Accessed 6 Nov 2001.

3. Machin NA, Lee JM, Barnes G.  Sept 1995.  Microtubule stability in budding yeast: characterization and dosage suppression of a benomyl-dependent tubulin mutant.  Mol Biol Cell. <http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&uid=8534919&dopt=r>  Accessed 6 Nov 2001.

 4. YRC Two-Hybrid Analysis.  2001. <http://depts.washington.edu/%7Eyeastrc/th_11.htm>  Accessed 6 Nov 2001.

5. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM.  2000. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.  Nature. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10688190&dopt=Abstract>  Accessed 6 Nov 2001.

6. Zhu H, Klemic JF, Swan Chang S, Bertone P, Casamayor , Klemic KG, Smith D, Gerstein M, Reed MA  and Snyder M. 2000. Analysis of yeast protein kinases using protein chips. Nature Genetics. <http://www.nature.com/cgi-taf/DynaPage.taf?file=/ng/journal/v26/n3/full/ng1100_283.html>  Accessed Nov 6 2001.

7. WIT Database. 2001. <http://wit.mcs.anl.gov/WIT2/CGI/index.cgi>  Accessed 6 Nov 2001.

8. EMP Database. 2001. <http://emp.mcs.anl.gov/cgi-bin/map_search.pl> Accessed 6 Nov 2001.

9. SGD. 2001. <http://genome-www4.stanford.edu/cgi-bin/SGD/locus.pl?locus=CKS1>.  Accessed 7 Nov 2001.

10. Schwikowski B, Uetz P, Fields S. 2000. A network of protein-protein interactions in yeast.  <http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11101803&dopt=Abstract>  Accessed 7 Nov 2001.

11. DT Jones. 1999. GenTHREADER: An efficient and reliable protein fold recognition method for genomic sequences.  <http://insulin.brunel.ac.uk/psipred/>  Accessed Nov 6 2001.


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