New & Noteworthy
Two Tales of Two Tails
July 22, 2015
Tails let animals sense and interact with their environment at a distance from their bodies. It turns out that some proteins use their “tails” in a similar way. Except they take it one step further.
Some mythical creatures have tails that coil around each other. Septin proteins are not all that different! Image via Wikimedia Commons
In addition to sensing the environment, they can also use their tails as sort of fishing poles to catch proteins they need to interact with. And they do this with great specificity…it is like having that perfect lure that always catches one type of fish.
A great example of this is the septin family of proteins which includes many members that are “tailed” proteins. Septins are highly conserved proteins that typically have a globular GTP-binding domain adjacent to an elongated C-terminal extension.
Septins form structures that act as the boundaries between different cellular parts. In budding yeast cells, they form the septum that separates the mother and bud, and recruit the cytokinesis machinery that allows the daughter cell to separate from the mother cell. In larger animals, they can be found in places such as the dendritic spines of neurons, sperm flagella, or cilia. And in humans, septin mutations have been linked to cancer and neurological diseases.
Until now, the details of how septins recruit other proteins to boundary sites have been elusive. But in two new papers in GENETICS, Finnigan and coworkers in the Thorner lab at Berkeley dove into this question and gained real insight into the lures these proteins use.
In their first paper they reported an extremely comprehensive genetic analysis to dissect the functions of two of the least characterized septins, Shs1 and Cdc11. In the second paper they used both genetic and physical methods to show how these septins recruit myosin to the septum to form the contractile ring that pinches off the bud from the mother.
The bottom line: their C-terminal tails are extremely important. They intertwine with other proteins’ tails like love-struck seahorses. And their specificity comes from these same tails—certain tails only coil around other tails.
The S. cerevisiae genome encodes a family of septins that assemble with each other to form octameric rods that consist of four different septins. The rods have both end-to-end and side-to-side interactions with each other, forming a ladder-like superstructure.
The septins Cdc11 and Shs1 are the most closely related members of the septin family, and the most recently evolved. They cap the ends of the septin rods. In otherwise wild-type cells, Cdc11 is essential for life while Shs1 is not.
Because SHS1 can be deleted without causing a major phenotype, the first step in investigating its function was to find genetic conditions under which its function becomes more obvious. The authors created four different genetic backgrounds in which the function of other septins was compromised by different mutations. Cells that had mutations in both SHS1 and in other septin genes had obvious problems, such as elongated buds or the inability to grow at high temperatures.
Now Finnigan and colleagues were set to do a detailed genetic analysis to figure out what different parts of Shs1 do by testing mutant versions in these different backgrounds. We can’t possibly recapitulate all the results here, but we’ll do our best to cover the highlights.
Almost all septins, whether in yeast or mammals, end with a tail: a long stretch called the C-terminal extension (CTE) that contains sequence patterns characteristic of a coiled-coil structure. The researchers found that the coiled coil regions of Shs1 and Cdc11were essential to their functions. (And no, they didn’t create any mutations by writing their names in the coiled coil sequence!)
Finnigan and colleagues tried swapping CTEs between different septins. When Cdc11 carried the Shs1 CTE and vice versa, the cells grew just fine. However, this swappability didn’t extend to other septins that are positioned internally in the septin rods. The CTEs of the end subunits Cdc11 and Shs1 could be exchanged for each other, but these CTEs only worked when they were on the ends of the rods.
Since coiled coils are often involved in interactions between proteins, Finnigan and colleagues wondered whether the essential function of the Cdc11 and Shs1 CTEs might be to recruit other proteins to the bud neck.
To test this, they searched published data to identify proteins that are well-known to be localized to the bud neck at the time in the cell cycle when septins are present. They found 30 such proteins, and overexpressed GFP-tagged versions of each in a strain where both Cdc11 and Shs1 lacked their CTEs.
Of the 30 proteins, only overexpressed Bni5 suppressed the growth defect of this strain. To test directly whether binding to Bni5 is a critical function of Shs1, Finnigan and colleagues fused the two genes to each other, so that Bni5 replaced the CTE of Shs1. This fusion protein could compensate for the lack of both Cdc11 and Shs1 CTEs.
To confirm that the important function of the CTEs is to hold Bni5 in the right place, they came up with an alternative test using a “nanobody”, which is a very small, very high-affinity single-chain antibody. They replaced the CTE of either Cdc11 or Shs1 with a nanobody that recognized GFP, and expressed a GFP-Bni5 fusion in these strains. In both cases, tethering Bni5 to the septin via the nanobody obviated the need for the CTE.
Finally, the authors asked why it is important for Bni5 to be located on the septin rods. Previous work had suggested that Bni5 recruits Myo1 (myosin), an important component of the contractile ring at the bud neck. They used the same nanobody constructs to test this, simply expressing GFP-Myo1 in the strains where the nanobody replaced the CTEs of Cdc11 or Shs1. Sure enough, tethering Myo1 to the terminal septins eliminated the need for Bni5.
So we now know that tails are absolutely essential for the functions of the alternative terminal septins Shs1 and Cdc11. These fishing poles let them hold on to the Bni5 “bait,” which in turn catches Myo1 to provide the muscle for cytokinesis to occur. Since septins are so highly conserved, it’s probable that these results will be directly applicable to higher organisms: there are mammalian septins that also occupy the end positions of septin rods, analogous to Cdc11 and Shs1. And that’s no fish story!
Not only septins use their tails to fish.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight