August 06, 2015
Some kids are born troublemakers, wreaking havoc and destruction everywhere they go. They can’t help themselves; it’s in their nature to be that way. But if they have concerned and protective adults in their lives, children can overcome this tendency and grow up to become productive members of society.
Within the cell, ribosomal proteins are problem children. Although they grow up to have essential and productive roles, as newborns they can cause big trouble.
Many of them have highly charged, unstructured regions that give them a tendency to aggregate with other proteins. And they have a complicated journey to adulthood, since ribosome assembly happens in multiple cellular compartments. With an estimated 160,000 ribosomal proteins synthesized every minute in rapidly growing S. cerevisiae cells, these troublemakers could cause major problems if left to their own devices.
To help control this unruly mob, certain proteins in the cell act as designated chaperones for ribosomal proteins. In a new paper in Nature Communications, Pausch and colleagues found that, surprisingly, these specialized nurses catch their client proteins even as they’re being born. They swaddle them from the first moment they start to emerge as nascent proteins, and keep them from causing any harm until they can be delivered safely to their final destination.
The researchers first looked at the proteins Rrb1 and Sqt1. Previous work had suggested they might act as specific chaperones for the ribosomal proteins Rpl3 and Rpl10, respectively. And Pausch and colleagues confirmed these results, showing that TAP-tagged Rrb1 pulled down only Rpl3 and Sqt1 only pulled down Rpl10. Each of these troublesome tots had its own personal chaperone!
But surprisingly, very little of the protein was needed for these chaperones to keep ahold of their respective charges. When the authors trimmed down the ribosomal proteins to shorter and shorter lengths, they saw that just the N-terminal 15-20 amino acids of each ribosomal protein were necessary and sufficient for interaction with its chaperone.
They decided to use X-ray crystallography to look in detail at the Sqt1-Rpl10 interaction. First they determined the crystal structure of Sqt1 on its own, and found that it forms an eight-bladed WD-repeat beta-propeller, looking much like a round electric fan. The amino acids positioned on the surface of the blades are negatively charged.
Next, the authors co-crystallized Sqt1 with a peptide corresponding to amino acids 2-15 of Rpl10. The structure showed that the positively charged peptide was cradled in the negatively charged surface.
To test whether these charged residues were important for the interaction, they mutated the charged residues of Sqt1 and of the peptide and combined them in various ways. Sure enough, changing the charged residues of either partner disrupted or diminished the interaction.
Pausch and colleagues went on to test whether those same charged residues are important in vivo. An sqt1 mutation changing glutamate residue 315 to lysine (E315K), that abolished the Sqt1-Rpl10 interaction in vitro, was lethal for yeast cells, confirming the importance of the interaction.
The researchers also detected many allele-specific genetic interactions between the charged residues of the two proteins, and even found that switching the charges in an interacting pair of amino acids (changing an Sqt1 residue to a positive charge and its Rpl10 binding partner to a negative charge) would improve growth compared to either single mutant.
The lethality of that sqt1-E315K mutation, and even the lethality of an sqt1 null mutation, were weakly suppressed by overproduction of Rpl10. So yeast cells can get by (just barely) with an un-chaperoned Rpl10, as long as there’s enough of it around. This result also confirmed that Rpl10 is the only client of Sqt1.
As yet another verification that Sqt1 acts as a chaperone, the authors looked to see what happens to the Rpl10 protein in sqt1 mutants. If cells carrying wild-type SQT1 are lysed and separated into a pellet and supernatant, most Rpl10 spins down in the pellet but a significant amount is soluble in the supernatant. However, if the cells carry any of several sqt1 mutant alleles that alter the charged residues and diminish the interaction with Rpl10, all of the Rpl10 is found glommed together in the pellet.
The two chaperone-ribosomal protein interactions that Pausch and colleagues investigated, Sqt1-Rpl10 and Rrb1-Rpl3, both involved the extreme N termini of the ribosomal proteins. Previous studies had also shown that two other chaperones for ribosomal proteins, Yar1 and Syo1, also interact with the N termini of their clients. So the authors wondered whether interactions between ribosomal proteins and their chaperones might even start during translation of the ribosomal proteins.
In a final experiment, the researchers treated yeast with cycloheximide to freeze translation and then pulled down each of the four chaperones via affinity tags. Each chaperone specifically pulled down the mRNA encoding its client protein, showing that it was binding to the nascent protein as it first started to emerge from the translating ribosome.
So this study has defined a new step in ribosomal biogenesis. Certain specific ribosomal proteins are such troublemakers that it’s too dangerous for the cell to just release them into the cytoplasm after they’re translated.
Instead, these bouncing baby proteins are caught by their individual nurses before they’re even fully born, and wrapped up to protect both the ribosomal proteins themselves and the rest of the cell. Since ribosomal biogenesis is highly conserved across species and since defects in it are associated with many different diseases, further study of these cellular midwives could have important implications for human health. Perhaps some gentle guidance could help put wayward ribosomes on the right track.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight