This molecular sculptor may have molded and re-formed the outer cell membrane of primitive bacteria into the intricate and interconnected protein transport network inside cells that is essential for the function of cells ranging from yeast to plants to humans.
This transportation network, called the endomembrane system, and the presence of a nucleus that contains the cell's DNA, are among the features that distinguish eukaryotic cells -- which make up plants, animals and humans -- from the prokaryotic cells that make up bacteria.
Reporting in PLoS Biology, Rout, together with Rockefeller colleague Brian T. Chait, D.Phil., and Andrej Sali, Ph.D., a former Rockefeller scientist now at University of California, San Francisco, describes a three-dimensional sketch of a core building block found in a structure called the nuclear pore complex (NPC) in eukaryotic cells. A massive complex of proteins, the NPC serves as a checkpoint between the cell's nucleus and the surrounding soupy cytoplasm inside the cell. Small molecules can enter and exit the nucleus through the NPC at will, but larger proteins and RNA molecules are allowed entry selectively.
"Buried in the NPC is a fossil indicating what it evolved from," says Rout, assistant professor and head of the Laboratory of Structural and Cell Biology at Rockefeller. "And we believe the job of this fossil is similar to its progenitor -- namely to curve membranes."
This discovery gives scientists a significant insight into the evolution of eukaryotes, or cells that carry their DNA inside a nucleus. "By manipulating, shaping and pulling in a membrane, cells could create tiny compartments that perform many of the jobs they need," Rout adds. "Thus, the eukaryotic cell freed itself from relying on only a cell wall for many of its membrane-associated functions, and allowed the cell to develop a host of intricate new internal processes."
Scientists believe the emergence of organelles, compartments in the eukaryotic cell's cytoplasm that perform such functions as energy production, waste removal and protein synthesis, and a nucleus evolved between 2 and 3 billion years ago.
One hypothesis regarding the evolution of eukaryotic cells suggests that the endomembrane system developed because some ancient bacterial cells had the ability to sharply curve their membranes, allowing them to form internal membrane structures as well as to engulf other organisms. The findings reported by Rout and colleagues suggest that an ancestor of an NPC component, called the Nup84 complex, may have been a key molecular sculptor responsible for such a reshaping of the membrane.
In 2000, Rout and Chait published the first complete inventory of the proteins in the yeast NPC. They showed that only 30 proteins, called nups, make up this structure. Since then, Rout and Chait have been collaborating with Sali to visualize what the NPC looks like, through a mathematical technique Sali pioneered called homology modeling. Because the NPC is difficult to crystallize, conventional protein imaging techniques, such as X-ray crystallography, cannot readily be used to solve the three-dimensional structure of this large molecular assembly in its entirety.
For the PLoS Biology paper, Rout and colleagues focused on a group of seven proteins that form the Nup84 complex in the yeast NPC. NPCs are uniformly located throughout the membrane that bounds the nucleus of a eukaryotic cell; they connect the inner and outer nuclear membranes through sharply curved sections of pore membranes. Comprising one-fifth of the whole NPC, the Nup84 complex is found where the curve of the pore membrane is sharpest.
Using a combination of Sali's mathematical homology modeling technique and biochemical analyses, the scientists -- led by first author Damien Davos, Ph.D., and Svetlana Dokudovskaya, Ph.D. -- produced three-dimensional models and found that the Nup84 complex in yeast is composed of two types of protein structures, "alpha solenoids" and "beta propellers." Two of the proteins are beta propellers, three are alpha solenoids and two are composed of beta propeller "heads" attached to alpha solenoid "tails." The scientists showed that the architecture of the Nup84 complex also appears in the NPCs of human and plant cells and is therefore conserved throughout eukaryotes.
Rout and colleagues extended this analysis and made an unexpected discovery: They showed that the Nup84 complex shares its structure with three major classes of molecules called "vesicle-coating complexes," which are responsible for shuttling membrane-bound packages to various sites in the cell.
The Rockefeller and USCF teams compared the alpha solenoid/beta propeller arrangement of the Nup84 complex with proteins from other organisms and found that only eukaryotes share this architecture. And the proteins currently known to contain this arrangement exist only as components of coated vesicle complexes or NPCs.
"The common architecture underlying the Nup84 complex and all three major classes of vesicle-coating complexes may be because all of the coating complexes have a common role in curving membranes," says Rout. "Moreover, we also believe these similarities indicate that NPCs and vesicle-coating complexes likely originated from a common ancestor."
The research published in PLoS Biology represents the first stage of an overall analysis of the NPC structure by the Rockefeller scientists.
"We can already see there will be more surprises like this in store," says Rout.