BOSTON--After ten years of trying to get his arms around his favorite molecule, Tom Rapoport is finally savoring an even better treat: He can lay his eyes on it, too. The professor of cell biology at Harvard Medical School has steadily inched toward an understanding of an all-important channel that is central to protein trafficking and secretion in cells of organisms ranging from bacteria to mammals. The existence of such a channel had been postulated 20 years ago as key to all normal cell function, yet it remained stubbornly elusive.
Finally, after years of amassing biochemical evidence that bolstered this prediction, the time was ripe to try to visualize the phantom molecule. In the November 15 Cell, Rapoport and his colleagues reveal the first, still blurry image of the membrane-spanning molecule.
The study sheds light on a fundamental process that occurs in all cells at every moment during an organism's life. It advances current understanding of how this unusual molecule works, and related research suggests that this molecule can be exploited by pathogens.
Mammalian cells use an elaborate transport system to ship proteins from their site of production, the cytoplasm, to their final destinations. Whether proteins end up being secreted, as is insulin, or inserted into the cell's membrane, as are receptors for growth factors, they all are sorted and delivered by the endoplasmic reticulum (ER), a meshwork of one continuous membrane sac that extends in tentacles throughout the cell. To enter the ER, proteins must cross its membrane, but membranes exist to prevent just that. So the question of exactly how a protein can overcome this barrier has spawned the lively research field of protein translocation.
Rapoport's earlier research had found the proteins of the Sec61p complex to be the most likely candidate molecules for the channel; his group even used the Sec61p complex to recreate protein translocation in artificial membranes outside of cells.
To visualize the Sec61p complex, Rapoport and his team collaborated with electron microscopy experts Ken Miller at Brown University and Christopher Akeys' group at Boston University. The researchers isolated the Sec61p proteins embedded in ER membranes and exposed the inside of the membrane by using an old technique called freeze fracture. When they peered through the electron microscope, they saw to their delight that what had 3felt and smelled2 like a channel indeed looked like one: tiny rings with a hole in the middle.
After analyzing the donut-shaped rings, the scientists integrated the
new and old data into a proposal for how protein translocation likely occurs.
When a ribosome making a new protein docks onto the ER membrane, four individual
Sec61p proteins Even more unusual, Rapoport says, is that the channel can open in two
dimensions to deliver secreted and membrane-bound proteins properly in different
ways. For example, insulin passes all the way through the ER membrane into the
lumen of the ER and eventually gets spilled into the bloodstream. However, the
insulin receptor slides only halfway into the ER membrane. Then the channel
opens sideways much like a handcuff to release the receptor into the ER membrane
from which it ultimately is pinched off in tiny vesicles that merge with the
outside cell membrane.
Protein translocation is so fundamental, it is very similar in all
living creatures. Even bacteria use a protein translocation system for
secretion. While there are no known diseases of protein translocation Tom Rapoport will be available for interviews this Wednesday and from Friday on.
A limited number of prints depicting electron-microscopic images are also
available.