Fast Tracking Protein Delivery into the Nucleus

Rush hour never ceases at the nucleus’ border. Gene products begin their lives in the nucleus as strands of mRNA that ship out into the cytoplasm, where they serve as templates for protein synthesis. Many of these proteins, such as transcription factors, subsequently return to sender, crossing back into the cell’s central organelle.¹ To traverse the nuclear envelope, proteins must transit through a nuclear pore complex (NPC).² This protein channel serves as a gatekeeper to the nucleus, restricting passage to select proteins that carry a nuclear localization signal (NLS)—an amino acid sequence that stamps the protein for delivery to the nucleus. 

In a recent study published in Nature Physics, researchers found that proteins harboring a flexible domain near the NLS enter the nucleus faster.³ To mimic this limber protein element, the biophysicists designed a bendable protein tag that expedited delivery of protein cargo into the cell’s core organelle. 

“People are certainly looking at how to deliver various therapeutics, diagnostics, or just simply research tools to the nucleus, and this could be rather important in very significantly improving the efficiency of that process,” said Michael Rout, a cell biologist at the Rockefeller University who was not involved with the work.

Scientists previously found that the NPC shapeshifts to allow cargo to cross the nuclear threshold, but scientists know little about how structural alterations to the protein parcels themselves affect transport.⁴ “Cargoes have been to some degree seen as like the corpse at a funeral—they’re the purpose for the whole ceremony, but they don’t take an active part in the process,” said Rout. 

To study the relationship between a protein’s molecular makeup and its movements, Sergi Garcia-Manyes, a biophysicist at the Francis Crick Institute and study coauthor, developed a system to time protein transport into the nucleus. He and his team chose a common protein motif called the immunoglobulin domain (Ig) as their test subject. They worked with two Ig mutants: one that was more flexible and another that was more rigid compared to wild type Ig. However, Ig domains don’t have an NLS, so the researchers gave each version the nuclear postage stamp. Fusing these mutant constructs to a fluorescent protein allowed the researchers to monitor protein distribution under a microscope and time their shipments to the nucleus. The researchers were ready to take their souped-up proteins to the races. When they pitted the mutants against one another, they found that the flexible Ig domain took less time to enter the nucleus than the stiff variety.

Garcia-Manyes and his team wondered whether the proximity of the flexible domain to the NLS affected transport speed. However, they could not use the Ig variants to test this hypothesis as the mutations were fixed near the NLS. Instead, they turned to the wild type version of Ig and attached a flexible protein called R16 to either end of its protein chain. By manipulating the distance between the flexible domain and the NLS, they determined that the closer the two elements were, the quicker the protein’s entry into the nucleus.

Garcia-Manyes and his colleagues considered the applications of their findings for turbocharging nuclear import. “What we thought is, ‘instead of just making use of the properties of the protein itself, let’s do something artificial—let’s design something through molecular engineering,’” he said. To create a synthetic flexible protein tag to place near the NLS of stiff proteins, they developed polymers of glycine (G) and serine (S)—two amino acids that scientists frequently use to fit nimble hinges on proteins.⁵ A single GS tag had a negligible effect on the nuclear import rate of Ig, and large tags carrying 25 copies of GS slowed down traffic. However, a Goldilocks’ range of two to four copies of GS boosted haulage across the NPC.

The synthetic tag halved the delivery time, but its impact varied. “For a very soft protein with a tag, you wouldn’t really see much of a difference, but if that protein is very stiff [then] you see a very strong effect,” said Rafael Tapia-Rojo, a biophysicist at King’s College London and study coauthor.

The biophysicists want to explore which nuclear proteins naturally evolved flexible regions to assist them in transit. For example, one nuclear protein called myocardin-related transcription factor A harbors many intrinsically disordered regions—amino acid chains that do not have a fixed structure—which may facilitate entry into the nucleus.⁶ “It would be very interesting to look at other native cargoes that appear to have these disordered regions in them next to the targeting sequence and see how generally this is used,” said Rout.

In future experiments, the team plans to test whether protein flexibility has a similar effect on the rate of nuclear export as well as transport across other pores on cytoplasmic compartments, such as energy-producing mitochondria. “Then we could begin to engineer different mechanical strategies to block or unblock transport across organelles,” Garcia-Manyes said.

References

1. Lu J, et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Commun Signal. 2021;19(1):60.
2. Paci G, et al. Cargo transport through the nuclear pore complex at a glance. J Cell Sci. 2021;134(2):jcs247874.
3. Panagaki F, et al. Structural anisotropy results in mechano-directional transport of proteins across nuclear pores. Nat Phys. 2024;20(7):1180-1193.
4. Hakhverdyan Z, et al. Dissecting the structural dynamics of the nuclear pore complex. Mol Cell. 2021;81(1):153-165.e7.
5. Van Rosmalen M, et al. Tuning the flexibility of glycine-serine linkers to allow rational design of multidomain proteins. Biochemistry. 2017;56(50):6565-6574.
6. Infante E, et al. The mechanical stability of proteins regulates their translocation rate into the cell nucleus. Nat Phys. 2019;15(9):973-981.