August 07, 2014
To mate, the yeast Saccharomyces cerevisiae needs to shmoo — to generate a projection that reaches out to a nearby yeast of the opposite sex, until the yeast cell is shaped like the Al Capp cartoon character. And to shmoo yeast needs, among other things, polyamines like spermidine.
Spermidine is important for one of the most interesting proteins in the world, the translation initiation and elongation factor eIF5A. Not only is this protein pretty much conserved in just about every living thing, but it is also the only protein to have the unique amino acid hypusine. And to make things even more fascinating, there are two other conserved proteins whose only job is to convert a single lysine residue of eIF5A into hypusine, using polyamines like spermidine. Simply mind boggling.
In a new study in GENETICS, Li and coworkers provide compelling evidence that spermidine is important in yeast shmooing because of its involvement in the hypusinylation of eIF5A. They also found that one reason eIF5A is so important in this process is that it is necessary for translating Bni1p, a formin needed to organize the actin cables of the shmoo. Without these actin cables, the shmoo can’t form.
It looks like yeast needs eIF5A to translate Bni1p because of the long stretches of prolines found in this protein. This suggests that like its bacterial ortholog EF-P, a key job for eIF5A is to help the cell deal with polyproline stretches in proteins.
To show this the researchers made a set of targeted mutations to check whether hypusinylation of eIF5A is necessary for shmooing. When they knocked out LIA1, one of the enzymes that uses spermidine to convert lysine to hypusine, the resulting yeast failed to shmoo. Since the only known target of the Lia1 protein is eIF5A, this suggests that hypusinylation of eIF5A is critical to its function in shmooing.
They also used temperature sensitive mutants of eIF5A to show that this gene (HYP2, also known as TIF51A) is involved in shmooing. At the nonpermissive temperature, only 7.7% of yeast with the less severe mutant allele, tif51A-1, shmooed, while none of the yeast with the more severe mutation, tif51A-3, were able to shmoo. These two results taken together establish the importance of eIF5A in shmooing.
Because eIF5A was known to be important for translating polyproline regions, the researchers looked for yeast proteins with such stretches, with the idea that their failure to be translated may be behind the need for eIF5A in shmooing. They found 549 such proteins, and a comparison of their Gene Ontology (GO) annotations showed four overrepresented categories including “mating projection” (shmoo). They focused on a protein from this group, Bni1p, because it was known to be involved in shmoo formation and it was one of only two proteins with ten or more prolines in a row.
Bni1p is important for organizing the actin cables that are needed to make a shmoo. Li and coworkers showed that the temperature sensitive mutants of eIF5A and bni1 mutants had similar phenotypes in terms of actin organization in the shmoo.
So the idea here is that yeast need eIF5A to shmoo because they need eIF5A to translate Bni1p, and Bni1p is needed to set up the actin framework of the shmoo. In this hypothesis, it is the indirect action of eIF5A that prevents the shmooing. To test this hypothesis, the authors generated a bni1 mutant that lacked the polyproline regions.
They compared the transcript levels of wild type BNI1 and the mutant lacking the polyproline stretches using RT-qPCR and found that the presence of eIF5A didn’t matter much. The transcript levels of the mutant and wild type BNI1 were pretty much the same.
It was a different story for the protein levels. Using Western blots Li and coworkers saw very little wild type Bni1p, but lots of the mutant protein. The yeast cells struggled to translate wild type Bni1p but had no trouble with the mutant. The easiest explanation is that eIF5A is needed to help the yeast translate polyproline regions of proteins, including Bni1p.
Finally, to confirm the eIF5A and Bni1p connection, they showed that additional Bni1p could partially overcome the shmoo defect of the temperature sensitive mutants of eIF5A. Since this suppression was only partial, and since the mutant phenotype of the eIF5A mutant is more severe than that of the bni1 mutant, there are probably other proteins involved in shmooing that require eIF5A for translation. Some likely candidates are those proteins containing polyproline stretches that are annotated to the GO term “mating projection”.
Although a connection between oddly-shaped yeast cells and human fertility and/or disease may not seem obvious, there might indeed be one. It turns out that eIF5A is so highly conserved that human eIF5A works just fine when expressed in yeast, and mammalian formins, like Bni1p, are also proline-rich. Formins are necessary for polarized growth, which is a feature of both reproductive cell and cancer cell growth, and spermidine is required for fertilization.
Hard to believe that yeast channeling a cartoon character can teach us so much about the most fascinating of proteins, eIF5A. And maybe even shed light on our own fertility.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight