Functional wiggle room

Sitting in seminars, I frequently hear interesting tidbits and details which contribute to a perspective that I think most biologists take for granted, but which is quite different from popular conceptions of what's going on in the cell. Recently, I heard a description of a fairly typical result of a yeast genetics experiment that reinforced for me how flexible protein function really is.

If you read internet discussions of evolution (especially discussions of whether the power of mutation and natural selection can account for the molecular structures of the cell), you can encounter debates over how frequently mutations are beneficial, or how likely it is, given a function, that a protein will evolve to have that function (discussed in the Behe and Snoke paper, which is actually in a professional journal), and more along those lines. Many biologists who work with genetically manipulable model organisms like yeast, fruit flies, and small nematodes (worms), would find that these debates often lack an intuitive perspective that comes from spending a lot of time making lots and lots of mutations in a gene or an organism and looking at the results.

In yeast, researchers have deleted every single gene in yeast (one at a time of course!) and now are working to delete every possible pair of genes. Many researchers have mutated nearly every residue in a protein and looked at the function of these mutations (look here for one example of many). People who do this kind of thing get feel for just how flexible and resilient protein function is.

Anyway, a recent speaker at a meeting I attended mentioned an interesting example of "multicopy suppression," which basically means this: you delete a gene (which gives you a yeast strain with a detectable defect), and then look for other genes that, when present at high levels, can suppress this defect. From these kinds of experiments, you can infer something about the function of the gene that suppresses the defect. There is a gene in yeast called KEX2, which produces a protein that resides in an internal cellular membrane (the Golgi). This protein's job is to cut other proteins at specific places; these cuts are important processing steps for proteins that are secreted from the cell, like alpha-factor (a yeast pheromone essential for mating - yes, yeast mate! try Google for more...). When you knock out KEX2, the yeast cells are defective in mating because alpha-factor is not correctly processed. There is no backup system, like a redundant duplicate gene - you just take out KEX2 and these cells cannot mate. (Actually, this is true only of those cells that secrete alpha-factor - the other yeast "gender" secretes the pheromone a-factor, which is not processed by Kex2 and these cells mate fine when you delete Kex2).

So here you have a protein with a very specific function, in a specific location - Kex2 resides in the Golgi and cleaves proteins very specifically after the double-amino acid sequences lysine-arginine or arginine-arginine. You knock it out, and its function is gone. But now here's the beauty of multi-copy suppression: you can take the protein Yps2, put multiple copies in a Kex-2-deleted yeast strain, and you overcome the mating defect. When I was new to yeast I thought these types of suppression screens were crazy - kind of like taking the spark plugs out of your car and seeing if you can make up for this by adding other parts at random (or even worse, taking away other parts at random! this works in yeast too but don't try it on your car...). But they often work spectacularly well, and this is a standard genetic technique.

What is Yps2? It's a protein that is anchored outside of the cell, to the cell wall - in other words, it's not in the same place as Kex2. Yps2 also cuts other proteins (it's a "protease", to use the jargon), but at less specific sites - it cuts after any lysine or arginine amino acid (unlike Kex2 which only cuts after the double-amino acid sequences mentioned above). Because of how this gene was discovered, it was thought that Yps2 and Kex2 had overlapping functions - that they both were involved in processing proteins like alpha-factor. The only problem is that Yps2 is located at the cell wall, not in the Golgi, and Yps2 is a more indiscriminate cutter. Furthermore, Yps2 could not overcome the mating defect of Kex2-deleted cells unless it was present in the cell at artificially high levels.

So what was interesting was that the speaker at the meeting presented evidence that Yps2 plays a role in cell-wall integrity, and seemed to suggest that the initial idea that Yps2 overlapped functionally with Kex2 was not correct. (By the way, these proteins do not appear to be closely related in terms of sequence similarity, and they belong to different protease classes - Kex2 is a serine protease, and Yps2 is an apartyl protease, for those interested in the technical details.) What this means is that when you knock out one protein (Kex2), another protein (Yps2) - in an entirely different cellular location, with a more indiscriminate cutting specificity - can be forced to adequately take over the function of the deleted protein.

This isn't a formal argument about evolutionary potential, but it just gives a hint of the wide functional flexibility that evolution has to work with. In this experiment researchers artificially kept Yps2 levels high to cover the defect, but protein expression levels can also be altered in nature through mutation. There are many, many more examples of this type of thing, and it is the intuition growing out of this background that explains why the yeast biologists I know give little credence to the claims of evolution's critics who argue that small, gradual mutations cannot eventually produce major innovation in the cell.