journal club

Sleuthing Intracellular Communications, Part 2

An in-depth look at how yeast cells organize their intracellular signaling cascades.

In the last post we introduced the paper Pheromone-Dependent Destruction of the Tec1 Transcription Factor is Required for MAP Kinase Signaling Specificity in Yeast. In other words, we are looking at how a yeast cell manages its internal messages. We learned that normally, yeast grow in their filamentous phase, but if you expose them to a pheromone, they’ll transition into a mating phase. We learned about tec1, a transcription factor for filamentation genes, and we took a quick look at Fig. 2a, where we saw that after yeast cells are exposed to the pheromone, tec1 is degraded.  

There’s another protein, fus3, involved in the pheromone response. If fus3 is missing (we knock genes out of genomes to study their roles all the time), the yeast doesn’t respond to the pheromone.  

With these two keys, we can begin to explore just how this system works. Tec1 degradation and fus3 are both necessary for yeast to respond to the pheromone. Why?  

How is Tec1 Degraded?  

Ubiquitination is a common culprit when it comes to breaking down proteins inside a cell. Ubiquitin is a protein that binds to other proteins to destroy them. They wanted to know if tec1 gets degraded this way, and used western blotting to figure it out.  

In a western blot, proteins are extracted from cells and then separated out on a gel. The protein bands are blotted off of the gel and onto a filter. The next step is to stain for the protein you are looking for – with proteins, this immunoblot will use labeled antibodies. The researchers used antibodies that bind to ubiquitin-tec1 conjugates. The gel is shown in Fig. 2b. In it, there is no band – and thus no conjugate – when there is no pheromone. Turn the pheromone on, however, and tec1 is ubiquinated; a band shows up on the blot. This, then, is the pathway of destruction.  

Does fus3 have anything to do with this?  

For that, we’ll have to look at Fig. 3. First, they engineered two yeast strains: a fus3 knockout (fus3Δ) and a fus3 mutant (fus3K42R). (You can read more about tools for engineering microbes in a previous post about building genetic toolkits.) A quick word on the naming of mutants: K42R is code for how the genetic mutation affects the amino acid sequence it codes for. K and R are both amino acid codes that stand for lysine (K) and arginine (R). Lysine is the 42nd amino acid in the fus3 protein, and apparently at the active site. When you mutate the gene so that lysine becomes an arginine, you mess up the activity of that protein.

This switch from a lysine to an arginine stops fus3 from phosphorylating other proteins. A phosphate group is like a post-it reading “kick me!” slapped on the back of an unsuspecting victim; a phosphorylated protein is tagged for destruction. Does fus3 mark tec1 this way?

Fig. 3 is another gel showing how much tec1 is around when you pull different strings. If you knock out fus3, tec1 sticks around, even when the pheromone arrives. If you mutate fus3 with this K42R mutation, tec1 can again go unscathed. We also see two other strains with tec1 mutations (mutations T273M and P274S). Here, two different sites are mutated, the 273rd and 274th amino acids in the tec1 gene. In Fig. 4, we see a map of the tec1 gene’s amino acids. Threonine (T) is the binding site for the phosphorlation, and its neighboring proline (P) interacts with this binding event. If you mutate either of these amino acids, phosphorylation cannot occur because the active site has been broken. And if phosphorylation can’t occur, tec1 is never degraded.   

Collectively, then, this data shows a phosphate group moving from fus3 to tec1. Soon after this transfer occurs, tec1 is destroyed.

This is reinforced in Fig. 4b. Three strains are exposed to pheromone, off and on. Here, a band denotes where a phosphorylation reaction occurs. Negative controls are important: one well shows fus3 in the mix, but with no tec1. No band to see here.

Next is a repeat demonstration on the active site of fus3. If we mix tec1 and fus3-K42R, does phosphorylation occur? No band, so no phosphorylation.

Finally, we add the wildtype (WT) fus3 protein and tec1. Here, when the pheromone is added, tec1 gets phosphorylated, which we can see since tagged phosphate shows up as a band on the blot.

We know that ubiquitin degrades phosphorylated proteins. One member of the yeast ubiquitin class is Dia2, and this large protein has subunits cdc53 and cdc34. Knockout dia2 or mutate those two sites, and tec1 no longer gets degraded after pheromone exposure. (You can check Fig. 5 to see this.)  

So to sum up so far, when they sense a pheromone, yeast cells degrade tec1. This stops filamentation and starts a transition into mating phase. In more detail, this requires:

  • Pheromone activates fus3 
  • Fus3 phosphorylates tec1 
  • Dia2 degrades the phosphorylated tec1 
  • Tec1’s filamentation genes are no longer expressed 

Tec1 is a transcription factor for filamentation genes, so its demise means less expression of those proteins.

Next, the researchers took a look at signaling specificity.  

Tec1 & Signaling Specificity

Fig. 6 explores the response of yeast cells with varying intracellular communication dynamics. Different combinations of genomes and plasmids allow us to observe when the yeast experiences crosstalk. 

The scientists have engineered a system that connects the filamentation response elements (FRE) to lacZ, a staple of molecular biology experiments that allows for an easy measure of whether the system is on or off. LacZ encodes β-galactosidase; measure β-gal, and you know how strongly the filamentation system is “on.”

So high β-gal means the filamentation response is high, and low β-gal means the filamentation system is muted. They use this system to test different knockdowns. They’ve knocked tec1 out of yeast strains, leave some as is (as a control; if you’re checking the figure, these are the uninduced gray bars) and expose some to pheromone (to test response; in the figure, these are the induced blue bars). Now tec1 degradation is necessary for the mating response. Pheromone exposure should control whether the mating response is on (blue) or off (gray), but that shouldn’t work unless you restore some of the pieces.

We can add missing elements back into the mix with a plasmid. Add tec1 back into the system, and the filamentation response is low, as expected.

But throw in a mutant tec1, and suddenly, there’s crosstalk. In systems turned on with pheromone, there’s filamentation going on. This is demonstrated in various ways throughout Fig. 6A-D, in which tec1 restored suppresses filamentation. But add a mutated tec1, fus3 knockdown, or dia2 knockdown, and you get crosstalk, resulting in more filamentation.

Fig. 6E is a final visualization of crosstalk. Yeast plated on a petri dish grows differently if it’s in mating of filamentous phase. Filamentation means that it’s digging deep into the agar and clinging to it; the mating phase is less invasive. By simply washing the agar under running water, you can tell what phase the yeast is in.  

Mating-phase yeast will wash off the plate, but filamentous-phase yeast will stick.  

This means that for the fus3-knockout strain in wedge #11, crosstalk occurs and a lighter color denotes filamentation. For the wildtype (in #5) or a strain in which tec1 was knocked out and then restored with a plasmid (#2), a darker color denotes a mating phase that was rinsed away.  

Conclusions 

This paper is about intracellular communications, including crosstalk and signaling specificity. Communication requires a signal and a response. Some signals are vague and induce lots of responses; others specifically induce a single one. Often, the line of communication follows a cascade, each response becoming a signal for the next step. Or in the case of today’s paper, communication is a web with the same proteins responding differently under different signals.

Crosstalk – when different signaling pathways share proteins, it’s possible for something to go wrong and a cell to respond to a signal with the wrong response.

In today’s paper, crosstalk occurred if a pheromone told the cell to respond with the mating pathway, but the cell responded with the filamentation pathway instead.  

Signaling specificity this refers to how exact a signal (like a pheromone) has on a cell response (like transitioning to mating phase).  

Yeast usually have high signaling specificity when it comes to responding to a pheromone. If yeast cells lose fus3, or mutate tec1 so that can’t be tagged for destruction, they lose specificity. When that happens, the signal causes a mixed response, and filamentation continues as strong as ever.  

There are lots of biochemical cascades that happen within a cell; the pathway we explored today is just one of them. The cell is a like a city, and must respond quickly to new information. Webs of proteins drive those processes.  

References

Bao, M. Z., Schwartz, M. A., Cantin, G. T., Yates, J. R., & Madhani, H. D. (2004). Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast. Cell, 119(7), 991–1000. https://doi.org/10.1016/j.cell.2004.11.052

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