Who would have thought a hundred years ago that a simple fungus contains high-tech security gates guarded by agents that authenticate cargo before letting it through? That’s what advances in imaging are allowing scientists to observe. We must be among the most privileged in history to witness the foundations of biological life coming into focus in all their glory!

You got a simplified glimpse of this gate 13 years ago in the film Unlocking the Mystery of Life. It’s that circular portal that allows the messenger RNA out into the cytoplasm. We’ve learned a lot since then. We know now that the nuclear pore complex (NPC) is one of the largest and most complex molecular machines in the cell. Each eukaryotic nucleus is studded with NPCs. They resemble portholes with nets, like basketball hoops, but much more sophisticated. They are the gateways for traffic in and out of the nucleus, but not just any molecule can pass through. Each macromolecule needs a ticket and an escort, represented by tags and accessory proteins that first authenticate the cargo then accompany it in or out.

New details of the NPC architecture came to light in a couple of papers in Science this month. Regarding these papers, Katharine S. Ullman and Maureen A. Powers state:

Nuclear pore complexes (NPCs), first observed by electron microscopy 65 years ago, mediate selective transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells. Although the exact size and protein composition of NPCs can vary between species, these massive and complex machines are highly conserved in their overall organization, which consists of multiple copies of ?30 nuclear pore proteins, or nucleoporins (Nups), in a symmetrical eightfold radial arrangement. Deciphering the structure of this immense complex has required ongoing multifaceted approaches. On page 106 and 56 in this issue, Chug et al. and Stuwe et al., respectively, have employed parallel approaches in very distant species and arrived at remarkably similar and informative structures of an essential subcomplex of the NPC.

What were the “very distant species” with conserved structures? The Chug team used a frog; the Stuwe team studied a thermophilic (heat-loving) fungus. Both teams looked at one element of the eight-fold radial structure at the central inner ring of the pore. They found that this domain has coils of protein that extend out into the pore and make contact with coils from the opposite side. These coils form a net that blocks entry unless the cargo has the right password, consisting of an “usher” of sorts called a nuclear transport receptor (NTR).

What’s emerging is a different model from the previously-held vision of pores that dilate and constrict to allow passage. “Instead they suggest a rigid pore in which flexible domains called FG repeats fill the channel and form a barrier that can be traversed by receptors that carry cargos across,” Valda Vinson explains in a brief summary of the papers in Science. FG repeats are so named for the phenylalanine-glycine amino acid patterns. “Interactions between NTRs and FG repeats are essential for facilitated translocation,” Chug’s team says.

NPCs grant free passage to small molecules but become increasingly restrictive as the size of the diffusing species approaches or exceeds a limit of ?30 kD in mass or 5 nm in diameter. This property is critical for keeping nuclear and cytoplasmic contents separate. NTRs, however, are not bound by this size limit. They mediate facilitated NPC passage and ferry cargoes up to the size of newly assembled ribosomal subunits (?25 nm in diameter) between the two compartments.

For size comparison, consider that the inner ring complex is 425 kilodaltons (kD) compared to the 30kD mass limit of unescorted cargo (one Dalton = one atomic mass unit). The specific order of amino acids in each Nup protein is critical to its structure and function. Nup62, for instance, requires “kinks” that fold it into the pore-traversing domain.

“The kink is stabilized by a network of highly conserved hydrophobic and hydrophilic contacts,” the researchers say. Indeed, many of the residues show “extreme evolutionary conservation” in animals as diverse as amoebas, fungi, and higher animals. That’s the only context in which Chug et al. mention evolution — the lack of it.

Stuwe’s team also mentioned evolution only in the context of conservation, except for a nonspecific comment in the opening paragraph:

One of the hallmarks of eukaryotic evolution is the enclosure of genetic information in the nucleus. The spatial segregation of replication and transcription in the nucleus from translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are massive transport channels that allow bidirectional macromolecular exchange across the nuclear envelope (NE) and thus function as key regulators of the flow of genetic information from DNA to RNA to protein.

You can find the word conserved or conservation 16 times in the paper. In one case, structural conservation was found despite low sequence conservation. Mutation experiments, however, usually broke the function or reduced it dramatically; some cases were lethal.

The nuclear pore complex, therefore, is a highly selective filter for macromolecules. Why does that matter? Chug et al. explain:

For transport selectivity, preventing the passage of unwanted material is as important as allowing NTR�cargo species to pass. This is straightforward to explain by an adaptive barrier that seals around a translocating species, particularly because cohesive FG domains readily assemble such self-sealing barriers in the form of FG hydrogels.

But if anything and everything can attach NTRs to let it pass through, selectivity is also lost. “A ‘general gate’ would therefore stand open at all times and consequently fail as a barrier,” they realize. How do the NTRs know which cargo is legitimate? This point is not clear from the papers and probably will require more analysis. It appears that multiple interactions with other nucleoporins (Nups) take part in discrimination against unwanted molecules. But the mechanism is general enough to allow the FG domains to rapidly facilitate passage of a wide range of cargo sizes.

In a news item from Rockefeller University, Professor Michael P. Rout sheds some light on the tradeoff between selectivity and speed:

“Usually, binding between traditionally folded proteins is a time consuming, cumbersome process, but because the FG Nups are unfolded, they are moving very quickly, very much like small molecules. This means their interaction is very quick,” explains Rout.

The disordered structure of the FG regions is critical to the speed of transport, allowing for quick loading and unloading of cargo-carrying transport factors. At the same time, because transport factors have multiple binding sites for FG Nups, they are the only proteins that can specifically interact with them — making transport both fast and specific.

So in this case, “fuzzy” interactions are an asset. Rout thinks that this is “the first case where the ‘fuzzy’ property of an interaction is a key part of its actual biological function.“

How fast are the NPCs? Chug et al. provide some details about the diffusion rate of large molecules through the pore:

Facilitated translocation is usually completed within 10ms [milliseconds].. NPCs can conduct ?1000 facilitated translocation events or a mass flow of ?100 MD [megadaltons] per pore per second, which further implies that NPCs can translocate numerous species in parallel.

With some 2,000 NPCs in a typical vertebrate cell (Wikipedia), that provides a huge capacity for facilitated, authenticated transport.

Not sufficiently impressed yet? Consider that the entire nuclear envelope breaks down at cell division, all the parts are duplicated (including all the genetic information, which is also error-checked), and the entire nucleus, NPCs and all, is reassembled in both daughter cells, ready to go sometimes in just minutes. That’s intelligent design!

Image credit: Blausen.com staff. “Blausen gallery 2014”. Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. (Own work) [CC BY 3.0], via Wikimedia Commons.