New & Noteworthy

Budding Yeast Diversifies its Phosphatase Portfolio

March 10, 2016


Putting all your eggs in one basket can be dangerous! So too can putting all your activity in a single protein. Image from Andrew McDowell via Flickr.

You’ve probably heard the old saying, “Don’t put all your eggs in one basket.” The idea of course is that the wise thing to do is to spread out your possessions so when something happens to one set, you still have the rest. (See what Homer and Marge Simpson think of this saying.)

If it really is wise to follow this saying, then according to the results of a new study just published in GENETICS by Kennedy and coworkers, the budding yeast S. cerevisiae is wiser than the fission yeast S. pombe. Well, at least as far as for one part of entry into mitosis.

To enter mitosis, every eukaryote tested so far needs to increase the activity of cyclin dependent kinase 1 (Cdk1). Dephosphorylation of a key tyrosine residue in Cdk1 is an important part of this increased activity.

One of the big players in this dephosphorylation is the phosphatase Cdc25 in S. pombe or Mih1p in S. cerevisiae. In fact, it is so important in S. pombe, that deleting it is lethal. These poor cells arrest in G2 and eventually die.

The same is not true for S. cerevisiae. Deleting MIH1 has only mild effects—a slight delay in entering mitosis and starting anaphase. The phosphorylation on the key tyrosine on Cdk1p, Y19, remains for a longer period of time in this strain, but does eventually clear, explaining the delayed mitotic entry.

One interpretation of this result is that S. cerevisiae has spread its Cdk1 phosphatase activity over multiple proteins. Knocking out MIH1 still leaves enough Cdk1 activity to allow the cell to enter mitosis, albeit more slowly.

One likely suspect in S. cerevisiae is Ptp1p. Previous work had shown that in S. pombe, Pyp3, the homologue of Ptp1p, can also dephosphorylate Cdk1-Y19.

Kennedy and coworkers found that deleting both MIH1 and PTP1 in S. cerevisiae had a more severe effect on mitotic entry and exit from anaphase compared to deleting only MIH1. In addition, the level of Y19 phosphorylation on Cdk1p remained for an even longer period in the mih1 ptp1 deletion strain. But it was still not lethal and the cells did eventually manage to pass through mitosis.

These results suggest there is still another player involved. The next suspect these researchers focused on was protein phosphatase 2A (PP2A). Previous work had shown that mutation of the B-regulatory subunits of PP2A, Cdc55p and Rts1p, both affect Cdk1p phosphorylation.

Because of the multiple routes by which PP2A can affect entry into mitosis, the authors designed an in vivo phosphatase assay to accurately measure the level of phosphorylation of Y19 of Cdk1p. The results of this assay suggested that PP2ARts1 and not PP2ACdc55 affected the phosphorylation state of Y19.

Kennedy and coworkers finally managed to kill off their yeast by deleting MIH1, PTP1, and PP2ARts1! They had finally found enough of this yeast’s phosphatase activity to mimic the effects of just Cdc25 in the fission yeast S. pombe.

Fission yeast keeps all of its Cdk1 phosphatase eggs in the same basket, while budding yeast has at least three different options. Image from websuccessteam.com.

Using immunopurified protein complexes, Kennedy and coworkers were able to show that both Mih1p and Ptp1p could dephosphorylate Y19 of Cdk1. They could not, however, see dephosphorylation by PP2ARts1. It could be that their in vitro assay did not detect it for this protein or that PP2ARts1 works on a different phosphatase that affects Cdk1.

Bottom line is that the budding yeast has evolved such that the phosphatase activity needed to enter mitosis is spread out over multiple proteins. The fission yeast evolved in a way that kept all of its phosphatase eggs in the same basket, Cdc25. We’ll let you decide which yeast you think is the wiser.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Cdk1 , mitosis , phosphatase , PP2A , redundant regulation

A Lot Hinges on Ndc80

December 04, 2014


The ability to fold tent poles can help you get to beautiful places, but the ability to fold the Ndc80 complex is essential to life.
Image by Maria Ly

A collapsible protein complex makes sure that dividing cells get the right number of chromosomes:

The invention of collapsible tent poles was a boon to backpackers everywhere. These long, rigid poles provide strong support for a tent, but when it’s time to pack up and go, they fold up into a short package. The secret? Flexible regions in between the rigid sections.

Although the inventors of these poles couldn’t have known it, something similar already existed in nature. In a new paper in GENETICS, Tien and colleagues used the awesome power of yeast genetics, along with some cool biochemistry, to look at the shape of the S. cerevisiae Ndc80 complex in vivo. They found that, just like a tent pole, it bends sharply at a flexible region to fold two rigid sections next to each other. And this ability to fold is critical for accurate chromosome segregation.

During cell division, chromosomes must be correctly attached to the mitotic spindle so that the mother and daughter cells each get one, and only one, copy of each. If a yeast cell doesn’t get this process right, it can become sick or even die. And if it happens in an animal cell, it can lead to cancer.

The Ndc80 complex is shaped something like a dumbbell.

The Ndc80 complex, which is conserved from yeast to humans, is an integral part of this process because it connects the chromosomes to the spindle during mitosis. It consists of four subunits and has an elongated middle and globular parts on each end, sort of like a dumbbell. The Ndc80 protein, one of the subunits, has an unstructured  “loop” region in the middle of its elongated section.

Previous work had shown that the loop region of Ndc80 is flexible in vitro, and in vivo experiments had shown that the whole complex can change its conformation. Tien and colleagues wanted to know whether the Ndc80 loop region was important for the shape of the complex during mitosis, and whether flexibility in this region was important for function.

They started with a genetic approach, and isolated mutations in NDC80 that caused heat sensitivity. One particular allele, ndc80-121, was especially interesting. The mutant protein had two amino acid changes, near each other and near the loop region. The Ndc80 complex containing the mutant protein was just as stable, and bound to microtubules just as tightly, as the wild-type complex. So why did the cells die at higher temperatures?

Tien and colleagues visualized mitosis in the mutant cells using fluorescence microscopy. They could see that when they raised the temperature, dividing mutant cells had lots of aberrant attachments between chromosomes and the spindle. Because of these attachments, proceeding through mitosis caused their spindles to break—a lethal event.

However, if they timed the temperature shift to happen later in the cell cycle, the ndc80-121 mutant cells were fine. If chromosomes had already been lined up correctly on the spindle before the temperature was raised, then the rest of mitosis could go on without a problem.

Tien and coworkers wondered whether the mutation might disrupt the binding of some other protein to the complex at high temperatures. To look for interactions, they selected mutations that suppressed the heat-sensitive phenotype of ndc80-121. But they didn’t find any suppressor mutations in other genes. However, they did find an intragenic suppressor mutation within the ndc80-121 gene.

Interestingly, this mutation affected a residue that was on the other side of the loop relative to the original two changes. If the Ndc80 complex is a dumbell, imagine that the dumbell is collapsible like a two-segment tent pole, with the loop region of Ndc80 as the elastic between the sections. If you folded the complex in this way, the amino acids changed in the ndc80-121 mutant protein would be positioned close to the amino acid that the suppressor mutation affected—an intriguing explanation for how these mutations might affect each other.

The flexible loop region of Ndc80 allows it to fold tightly, like a tent pole.

Of course, genetic interactions don’t prove a direct physical interaction. So the researchers looked to see whether they could detect physical interactions between these regions. They treated the complex with a reagent that would permanently cross-link amino acids that were close to each other. Then they chopped the complex into smaller peptides using a protease, and analyzed the cross-linked peptides using mass spectrometry to locate the linked residues.

Sure enough, they were able to detect multiple cross-links within the complex, and their locations confirmed that the complex folds much like a tent pole. Based on their mutant phenotypes, the researchers think it’s likely that the original ndc80-121mutation destabilizes folding of the complex and that the intragenic suppressor mutation makes folding tighter. Consistent with this idea, the intragenic suppressor mutation alone confers a slow-growth phenotype, as if it makes the complex fold just a little too tightly to support vigorous growth.

These experiments as a whole establish that the Ndc80 complex folds tightly early in mitosis. So, creative inventors and Mother Nature have arrived at similar solutions for the tent pole and for this important complex. And just as collapsible tent poles have become ubiquitous in the backpacking world, so too has the collapsible Ndc80 complex been conserved throughout evolution: even the specific residues that mediate the folding are highly conserved. Since this work has shown that correct folding of the yeast complex is necessary for its role in helping chromosomes to line up accurately on the spindle, the same is almost certainly true in mammalian cells.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: protein structure , mitosis , Ndc80 complex , Saccharomyces cerevisiae

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