May 7, 2004

Key to the portal: a fresh look at in-cell transport

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Susan R. Wente, Ph.D.

Key to the portal: a fresh look at in-cell transport

The ability to shuttle proteins and genetic material between the nucleus and the cytoplasm is essential to the proper functioning of the cell, yet this process is poorly understood.

In an elegant set of experiments, researchers led by Susan R. Wente, Ph.D., professor and chair of the Department of Cell and Developmental Biology at Vanderbilt University Medical Center, have teased out key operating elements of this shuttling process in yeast, and in so doing have challenged current thinking about how transport occurs.

Their discoveries were reported in the March issue of the journal Nature Cell Biology.

At the center of the transport mechanism are the nuclear pore complexes, protein “machines” that are embedded in the nuclear envelope separating the nucleus from the cytoplasm. Macromolecules such as proteins and RNA don’t just flow through the pores. They must be escorted by “transport factors” that bind to them and to the complexes in a precise series of steps.

Recent advances in genomics and proteomics have allowed scientists to begin to deduce how nuclear pore complexes work. The filamentous and ring-like structure of the nuclear pore complex in budding yeast and vertebrates is constructed from 30 different proteins, each of which is present at least eight times in terms of the overall symmetry. Approximately one third of these proteins contain distinct stretches of amino-acid sequences — called “FG domains” — that bind to specific transport factors on their journey through the pore.

There are several theories for how proteins and RNA are escorted through the nuclear pore complexes, but most of the models have assumed there was only one way through and only one universal mechanism. Wente and her colleagues found there are at least two pathways through the pore complex, “and that’s being conservative,” she said.

Using a strategy to specifically knock out only the portion of the genes that encode specific FG domains within nuclear pore complex proteins, and genetic techniques to engineer mutant yeast strains with minimal subsets of the FG domains, the researchers found that they could delete half of the domains, and half of their total mass, without affecting cellular function. They also discovered that a specific combination of domains was necessary for one kind of transport factor to “truck” its cargo across the membrane, whereas another transport factor required another combination.

These discoveries have shed light on the remarkable selectivity of nuclear pore complexes. “There might exist functionally independent routes through the (complex), potentially using the same innate mechanisms of translocation, but through different binding sites,” wrote Benjamin L. Timney, Ph.D., and Michael P. Rout, Ph.D., of the Laboratory of Cellular and Structural Biology at The Rockefeller University in New York, in a commentary that accompanied the Wente group’s paper.

The presence of more than one pathway is a good thing, Wente added. This work may provide future clues for ways to block pathogens such as the human immunodeficiency virus (HIV) from slipping their genetic material into the nucleus — without disrupting normal cell functions. Other signaling factors, such as those that trigger cancer cell growth or a “toxic shock” immune response to infection, also potentially could be specifically locked out of the nucleus.

“It’s basic cell biology,” Wente said. “If you want to try to inhibit something from moving from the nucleus to the cytoplasm (and vice versa), this is the portal.”

Wente’s colleagues in this study included her former graduate student Lisa A. Strawn, Ph.D., and former research associate Tianxiang Shen, M.S.; and Nataliya Shulga, Ph.D., and David S. Goldfarb, Ph.D., both in the department of Biology at the University of Rochester.

The study was supported by the National Institutes of Health.