Under The Hood Of A Cellular Transport Machine

Boston, MA (June 12, 1999) -- A collaboration between researchers in Great Britain, Italy, and Harvard has developed a newly detailed view of one of the cell's major transport vehicles responsible for shuttling into the cell a range of important molecules, including cholesterol.

The findings, presented in the June Molecular Cell, provide insight into how this machine, the clathrin-coated vesicle, is formed, stays together, and falls apart.

A video of the clathrin protein can be viewed on the Web at www.hms.harvard.edu/news/clathrin/. Clathrin-coated vesicles are constantly assembling and disassembling to perform their task of transporting proteins from the outside of the cell inside. They are responsible for importing LDL cholesterol, and they play a role in breast cancer through internalization of a key receptor.

During disease progression of HIV infection, clathrin-coated vesicles are subverted by a viral protein to cause down-regulation of the viral receptor CD4 in an important but not fully understood step. These molecules, and a wide range of others, are selectively trapped in the clathrin-coated vesicle for import into the cell.

The new insights into how the vesicle forms help build a picture of the overall process and suggest possible targets for future therapeutic intervention.

One mystery of clathrin vesicles is how the outer cage of clathrin assembles so rapidly. Vesicles are incessantly assembled and disassembled at an incredible scale. In the brain, where neurotransmitters are constantly released into synapses, the membrane used to export the neurotransmitters is constantly being dragged back in by clathrin-coated vesicles.

"The equivalent of the entire brain, or a football field of membrane, is turned over every hour," says Tomas Kirchausen, associate professor of cell biology at the Center for Blood Research and Harvard Medical School and senior author on the article last year describing clathrin's atomic structure.

The new work allows Kirchhausen and colleagues to propose that clathrin molecules add to the growing cage lattice by hooking into spaces in the existing structure, then rapidly rotating into a locked position. The process is reminiscent of images of alien space ships locking into the mother vessel (see video).

The new insights come from combining an overall view of a barrel-shaped clathrin lattice, obtained using cryo-electron microscopy by Barbara Pearse and colleagues at the MRC Laboratory for Molecular Biology in England, with the much more detailed view of a portion of the protein derived from X-ray crystallography by Tomas Kirchhausen, Stephen Harrison, and colleagues at Harvard Medical School, the Center for Blood Research, Children's Hospital and the Howard Hughes Medical Institute, and Andrea Musacchio, now at the European Institute of Oncology in Milan, Italy.

Both sets of data were published late last year, but by collaborating to combine them the researchers managed to identify the portions of the clathrin molecule that flex during the assembly of the lattice.

A single clathrin molecule is made up of three clathrin heavy chains combined at a hub to make a three-legged pinwheel, a triskelion. Each leg has a "knee" where the molecule bends and a "foot" that interacts with adapter proteins to form the lattice.

To bring something from the outside of the cell inside, clathrin molecules combine at the cell membrane to form a clathrin-coated pit on the inside surface of the outer cell membrane. The pit then round