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

The Story of a Yeast Receptor Gene and an Unknown Neighbor

PART 2: YEAST EXPRESSION

Note: This is the second part of a three part exploration.  To see information regarding these two genes, please go here.


Annotated Gene:  STE2 (YFL026W)

As a quick summary, the main purpose of this page is to explore the expression data regarding two yeast genes: STE2 and a possible gene known as BUD27.

Below is the gene ontology for STE2 (Cherry et al., 1997):

Molecular Function: mating-type alpha-factor pheromone receptor (Burkholder AC and Hartwell LH, 1985)

Biological Process:  pheromone response

Cellular Component:  integral plasma membrane protein

BUD27 is not annotated, so the gene ontology information is not available. Exploring possibilities for the gene ontology is the primary goal of this webpage.


EXPRESSION CONNECTION

One of the databases used to look at the expression profiles of STE2 and BUD27 was Expression Connection. This website, maintained by the Saccharomyces cerevisiae Database, includes information from microarray data. The expression of yeast genes in "wildtype conditions" was compared with the expression of genes in each of eleven experiements. On this page, only a selection of this data will be included. It is important to note that my gene in question (STE2 or BUD27) will be listed at the top of each data set, with the 20 genes that have similar expression ratios listed below the gene. In this way, we are able to see what genes may be regulated together or what conditions induce or repress certain sets of genes. Ideally, the data obtained will aid in determining the function of BUD27. First, however, we will look at expression data for STE2. Use the scale below to see and compare the intensities of the red and green colors to see the fold repression (green) or induction (red) from wildtype cells. (Also note, that at times, the experimental conditions are compared to other conditions noted and not necessarily to wildtype "control" conditions).

Scale : (fold repression/induction)

Click on a color strip to see data for that gene.


 

Expression at Different Alpha-Factor Concentrations for STE2/YFL026W (from Rosetta Inpharmatics; Roberts, et al., 2000)

 

Orf

 

Gene

 

 

Process

 

Function

 

Component

YFL026W

 

STE2

 

 

pheromone response (sensu Saccharomyces)

 

mating-type alpha-factor pheromone receptor

 

integral plasma membrane protein

YLR452C

 

SST2

 

 

signal transduction*

 

GTPase activator

 

plasma membrane

YBR083W

 

TEC1

 

 

pseudohyphal growth

 

specific RNA polymerase II transcription factor

 

nucleus

YBR065C

 

ECM2

 

 

cell wall organization and biogenesis

 

molecular_function unknown

 

cellular_component unknown

YNR001C

 

CIT1

 

 

tricarboxylic acid cycle*

 

citrate (SI)-synthase

 

mitochondrion*

YBL016W

 

FUS3

 

 

protein amino acid phosphorylation*

 

MAP kinase

 

cytoplasm*

YNR044W

 

AGA1

 

 

agglutination (sensu Saccharomyces)

 

cell adhesion receptor

 

cell wall

YPR122W

 

AXL1

 

 

bud site selection*

 

metalloendopeptidase

 

integral membrane protein*

YNL078W

 

NIS1

 

 

regulation of mitosis

 

molecular_function unknown

 

nucleus*

YHR018C

 

ARG4

 

 

arginine biosynthesis

 

argininosuccinate lyase

 

cytosol

YCL027W

 

FUS1

 

 

mating (sensu Saccharomyces)

 

molecular_function unknown

 

plasma membrane

YHR005C

 

GPA1

 

 

signal transduction of mating signal (sensu Saccharomyces)

 

heterotrimeric G-protein GTPase

 

plasma membrane*

YOL104C

 

NDJ1

 

 

synapsis

 

molecular_function unknown

 

telomere

YMR222C

 

FSH2

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YFL027C

 

GYP8

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YCL019W

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YNL201C

 

PSY2

 

 

biological_process unknown

 

molecular_function unknown

 

cellular_component unknown

YOL105C

 

WSC3

 

 

cell wall organization and biogenesis*

 

molecular_function unknown

 

membrane fraction

YLR042C

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

cell wall (sensu Fungi)

YPL058C

 

PDR12

 

 

transport*

 

xenobiotic-transporting ATPase

 

plasma membrane

YIL080W

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated


* : indicates that more than one annotation exists for the gene.

STE2 is the alpha-factor pheromone receptor located in the cellular membrane of mating type a cells. You can clearly see from the above data that as the concentration of alpha-factor increases, the expression of the alpha-factor receptor (STE2) increases. This makes sense if the cell senses more alpha-factor, it would want to make more alpha-factor receptors in order to bind to the alpha-pheromone so that the cell can undergo the mating reaction. Other genes that produce proteins that reside in the plasma membrane are also induced at this time, suggesting that all of these genes are regulated by some transcription factor that responds to alpha-pheromone. The next gene on the list is SST2 and it is a protein involved in the desensitization to alpha pheromone. It's actually involved in signal transduction of the alpha-factor signal (once it binds to the alpha-factor receptor).

Click on any of the above genes to see what each one does. This is representative of how interactive the Expression Connection website is. The table below is another example. However, every other shot on this page is not interactive. The other shots are merely screen shots taken from the website. The graph below is also a screen shot; thus its "links" are merely copies of what they look like on the original website. They do not work. The graph does, however, show how the expression of STE2 is induced and stays induced as the concentration of alpha factor increases.


Expression in response to alpha-factor  (Rosetta Inpharmatics; Roberts, et al., 2000)

Orf

 

Gene

 

 

Process

 

Function

 

Component

YFL026W

 

STE2

 

 

pheromone response (sensu Saccharomyces)

 

mating-type alpha-factor pheromone receptor

 

integral plasma membrane protein

YJL157C

 

FAR1

 

 

cell cycle arrest

 

cyclin-dependent protein kinase inhibitor

 

cytoplasm*

YML047C

 

PRM6

 

 

mating (sensu Saccharomyces)

 

molecular_function unknown

 

integral membrane protein

YFL027C

 

GYP8

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YGL162W

 

SUT1

 

 

regulation of transcription from Pol II promoter*

 

specific RNA polymerase II transcription factor

 

nucleus

YPR122W

 

AXL1

 

 

bud site selection*

 

metalloendopeptidase

 

integral membrane protein*

YML046W

 

PRP39

 

 

mRNA splicing

 

not yet annotated

 

commitment complex*

YNL145W

 

MFA2

 

 

mating (sensu Saccharomyces)

 

pheromone

 

extracellular

YHR142W

 

CHS7

 

 

ER to Golgi transport*

 

molecular_function unknown

 

endoplasmic reticulum membrane

YOR212W

 

STE4

 

 

signal transduction of mating signal (sensu Saccharomyces)

 

heterotrimeric G-protein GTPase

 

plasma membrane*

YCL027W

 

FUS1

 

 

mating (sensu Saccharomyces)

 

molecular_function unknown

 

plasma membrane

YBL016W

 

FUS3

 

 

protein amino acid phosphorylation*

 

MAP kinase

 

cytoplasm*

YLR433C

 

CNA1

 

 

cell wall organization and biogenesis*

 

calcium-dependent protein serine/threonine phosphatase

 

cytoplasm

YLR452C

 

SST2

 

 

signal transduction*

 

GTPase activator

 

plasma membrane

YBR133C

 

HSL7

 

 

regulation of cell cycle*

 

protein-arginine N-methyltransferase

 

bud neck

YBR156C

 

SLI15

 

 

chromosome segregation

 

protein kinase

 

kinetochore microtubule*

YHR005C

 

GPA1

 

 

signal transduction of mating signal (sensu Saccharomyces)

 

heterotrimeric G-protein GTPase

 

plasma membrane*

YNL123W

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YPL177C

 

CUP9

 

 

transcription initiation from Pol II promoter*

 

specific RNA polymerase II transcription factor

 

nucleus

YNL279W

 

PRM1

 

 

cell-cell fusion

 

molecular_function unknown

 

integral membrane protein*

YDR461W

 

MFA1

 

 

signal transduction of mating signal (sensu Saccharomyces)

 

pheromone

 

extracellular

In this table, the expression ratios of genes were determined as the cells were exposed to a set concentration of alpha-factor over time. Again, it is clear that the expression of STE2 and other genes associated with the signal transduction cascade in response to alpha-factor is increased as the cells spend more time in the presence of alpha-factor. The graph below (again, merely a screen shot) illustrates the trend that as long as alpha-factor is present, there will be an induction of STE2.


Expression during the diauxic shift  (Stanford University; DeRisi et al., 1997)

This table shows the expression of particular genes when the cells are moved from anaerobic to aerobic conditions (the diauxic shift). Over time, STE2 is repressed, but it takes 18.5 hours under aerobic conditions for this to occur. When such a major change comes over the yeast cells, there will always be a huge shift in expression levels of many genes. As it appears, many different kinds of genes follow the same trend as STE2. It does not appear to be specific to alpha-factor signal transduction genes as you can see upon first glance of the 20 genes with similar expression ratios to STE2.


Expression during sporulation  (UCSF, Stanford University; Chu, et al., 1998)

Under nitrogen starvation, diploid cells undergo meiosis and sporulation to form four spores. When yeast are starved of nitrogen, there is an immediate repression of STE2. Obviously, if cells are undergoing sporulation, then they are not going to be mating or responding to alpha-factor any time soon. There is not enough nitrogen for the mating process to occur, and thus there is no need for the alpha-factor pheromone receptor.


FUNCTION JUNCTION

Function Junction is another website used to assess the function of yeast genes. The database includes information gathered from six other databases. For STE2, we are not questioning its function, but merely illustrating that what we know about it corresponds to the information obtained from these databases. Each database does say that STE2 encodes the alpha-factor pheromone receptor and that that receptor is a 7-transmembrane protein embedded in the plasma membrane. The best database that is searched using Function Junction is the Yeast Microarray Global Viewer from the Laboratoire de genetique moleculaire, Paris, France. Function Junction takes a selection of these microarrays and shows how your specific gene is expressed under certain conditions.


Non-Annotated Gene: BUD27 (YFL023W)

BUD27 is not annotated, so the gene ontology information is not available. Exploring possibilities for the gene ontology is the primary goal of this webpage.

I first viewed BUD27 microarray data from Expression Connection. The same scale from above can be used and the type of information provided here about BUD27 is very similar to that provided about STE2.


Expression at Different Alpha-Factor Concentrations for BUD27 (from Rosetta Inpharmatics; Roberts, et al., 2000)

The known gene closest to BUD27 (there are four that are unknown) is a protein serine/threonine phosphatase that is responsible for the G1/S transition of the cell cycle. But this data does not appear to be significant. It is not induced nor repressed at increasing amounts of alpha factor. What is significant is that it does NOT respond the same way that genes involved in the alpha-factor signal transduction cascade respond, suggesting that it is not regulated nor involved with genes inolved in this signal pathway. This would make sense since the prediction is that BUD27 is involved in bud site selection. If a yeast cell is budding, then there must not be anyone around it to mate with. This information could also suggest that because the cell is getting ready to mate, it does not need to prepare to bud. Another interesting point about this data is that the BUD27 gene is not repressed. One would think that it would be repressed if it's not needed since the cell thinks it is about to mate.


Expression in response to alpha-factor  (Rosetta Inpharmatics; Roberts, et al., 2000)

Again, nothing significant occurs with BUD27 as the yeast cells are exposed to alpha factor for an increasing amount of time. This correlates with the data from the first alpha-factor experiment above. BUD27 most likely has nothing to do with the mating signal cascade.


Expression in response to DNA-damaging agents  (Stanford University; Gasch, et al., 2001)

This microarray looks at numerous DNA-damaging conditions. In most instances, where there is any change in expression for BUD27, it is a repression. The first set of data are the expression ratios of wildtype cells compared with wildtype cells exposed to methyl-methanesulfonate (MMS), a chemical DNA-damaging agent, for different lengths of time. The next set is wildtype cells compared with mec1 mutants that were exposed to MMS for different lengths of time. Mec1 is a DNA repair gene. Thus, if DNA is being damaged and it can't be repaired, one would expect a different set of expression ratios compared to wildtype DNA that still has its DNA repair gene. And in fact, that is what we see. BUD27 is repressed when wildtype cells are exposed to MMS but not when mec1 deletants are exposed. In the wildtype cells, MEC1 is still present and is able to recognize the DNA damage and tell the cell about that damage. There's nothing telling the cell that DNA is damaged in the mec1 deletants, so they continue with their "wildtype" activities and do not alter the expression of some of their genes. Also, in each set of conditions, BUD27 acts similarly to RNA binding genes and some other ribosomal genes, such as ones encoding RNA helicases..


Expression during the diauxic shift  (Stanford University; DeRisi et al., 1997)

 

When the yeast cells are moved from anaerobic to aerobic conditions, the expression of BUD27 appears to be induced at first, until about 13.5 hours, where it slips into repression. It reacts very similarly to other ribosomal or rRNA genes like SQT1 and SAS10. However, it acts most closely to genes of unknown function. Could this be a new class of genes?


Expression Regulated by the PHO pathway  (Stanford University; Ogawa et al., 2000)

A low phosphate concentration in the medium makes yeast induce a set of genes to adapt to the low-phosphate environment. This gene expression regulation is known as the "PHO" regulatory system. Thus, the genes above are repressed when phosphate is limited. BUD27 acts like genes involved in 35S primary transcript processing and some rRNA modification. Again, BUD27 could have processing or regulatory roles involved transcripts in yeast. Unfortunately though, the genes closest to it in expression ratios are not annotated genes. It is interesting to note that so far, no other "BUD" genes have been observed on any of these microarrays. This fact still leads me to believe that BUD27 is a transcriptional regulator of those genes responsible for budding, instead of those genes themselves.


Expression during sporulation  (UCSF, Stanford University; Chu, et al., 1998)

As the cells are deprived of nitrogen over time, they undergo sporulation. As time goes on, BUD27 is induced. If BUD27 is indeed involved in some sort of budding or sporulation mechanism (two processes that are very similar and could use the same set of genes) then this data would support that. In fact, BUD27 acts more similarly to transcription regulators like CHA4. This correlates with information obtained previously about the gene in general. Maybe BUD27 is a transcription factor used to regulate other genes involved in the budding and sporulation processes.


FUNCTION JUNCTION

The information below was obtained from Function Junction.

Yeast PathCalling Home Page (CuraGen & University of Washington)

The figure above shows other gene products that BUD27 (represented as YFL023W here) might interact with. The two genes immediately connected toYFL023W are RPB5 and YKE2. YKE2 is located in the cytoplasm and is involved with tubulin and protein folding. RPB5 is part of the DNA-directed RNA polymerase complex and is involved in transcription. If these two gene products interact with BUD27, then RPB5 could be the transcription factor that helps transcribe BUD27 and YKE2 could be a gene product that BUD27 acts on. If BUD27 is involves with budding, then it would make sense for it to act on or interact with a gene that is involved with tubulin and protein folding. Conversely, the protein encoded by BUD27 could be folded by YKE2, suggesting its possible tubulin properties? A closer look reveals that GIM5 is also a protein involved in protein folding. However, FAR3 is a protein needed to arrest the cells in G1 of the cell cycle in response to the mating pheromone. By looking at the microarray data above, I had ruled out BUD27's role in pheromone response or in mating. Perhaps, however, the interaction between BUD27 and FAR3 is a "negative" interaction. Maybe BUD27 is responsible for turning FAR3 "off." If BUD27 is a transcriptional regulator or transcript processor, then this suggestion would work.

Conclusion

In lieu of this data, I believe that BUD27 is a transciptional regulator or processor that interacts with genes and/or proteins involved in the budding pathway. It could also act to turn off genes/proteins that may be involved in the mating pathway so the cell does not prepare itself for mating, but redirects its energy to the budding pathway.

References

Burkholder AC and Hartwell LH. 1985. The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res 13(23):8463-75.

Cherry, J. M., Ball, C., Dolinski, K., Dwight, S., Harris, M., Matese, J. C., Sherlock, G., Binkley, G., Jin, H., Weng, S., and Botstein, D. 1997-2000. "Saccharomyces Genome Database" http://genome-www.stanford.edu/Saccharomyces/ Accessed: 04 October 2002. *Function Junction and Expression Connection are interactive databases associated with SGD.

Chu S, et al. 1998. The transcriptional program of sporulation in budding yeast. Science 282(5389):699-705. (abstract)

DeRisi JL, et al. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278(5338):680-6. (abstract)

Gasch AP, et al. 2001. Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p. Mol Biol Cell 12(10):2987-3003. (paper)

Ni L and Snyder M.  2001. A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae. Mol Biol Cell 12(7):2147-70 (paper)

Ogawa N, et al. 2000. New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis. Mol Biol Cell 11(12):4309-21. (paper)

Roberts CJ, et al. 2000. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287(5454):873-80 (abstract)

Sprague GF.  1991.  Signal transduction in yeast mating: receptors, transcription factors, and the kinase connection.  Trends Genetics 7(11-12):393-8.

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