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U-M scientists see ubiquitin-modified proteins in living cells

11.10.2004


New technology makes visualization possible



Researchers at the University of Michigan Medical School and Howard Hughes Medical Institute have found a way to see proteins in cells that have been tagged by a molecular "sticky note" called ubiquitin. "This technology allows us to see, under a microscope, proteins modified by ubiquitin inside the cell," says Tom K. Kerppola, Ph.D., an associate professor of biological chemistry in the Medical School and an HHMI associate investigator. "Visualization gives us a direct connection to cellular processes that we could only study in test tubes or indirectly before."

In a paper published online this week in the early edition of the Proceedings of the National Academy of Sciences, Kerppola and Deyu Fang, M.D., Ph.D., a U-M research investigator, describe the first use of a technology called ubiquitin-mediated fluorescence complementation to study a cell-signaling mechanism called ubiquitination.


In this process, a small peptide called ubiquitin is linked to a protein in ways that can change the protein’s function and location within the cell. Originally, scientists thought ubiquitin was simply a universal "destroy me" signal for unneeded or harmful proteins, but it has recently been found to be associated with many other cellular functions. "The same ubiquitin signal can cause one protein to be degraded, but another protein to be moved to a new location," Kerppola says. "We’re interested in learning how this works."

In their PNAS paper, Kerppola and Fang describe how ubiquitin latched onto Jun - a protein involved in cell growth and gene transcription – and moved Jun from its usual location in the cell’s nucleus into hollow spheres called lysosomes in the cytoplasm outside the nucleus. Filled with digestive enzymes, lysosomes break down unwanted proteins into amino acids the cell recycles to make new proteins. "Jun’s function in the nucleus is transient and time-dependent," Kerppola says. "If it’s turned on and doesn’t get turned off, that’s not normal. Prolonged signaling causes aberrant growth and other problems for the cell."

"When ubiquitin is attached to Jun, the complex is transported to the lysosome getting it away from DNA in the nucleus and preventing Jun from continuing its normal gene transcription function," he adds. "This is a new way for the cell to eliminate the function of a transcription factor."

U-M scientists also discovered that an E3 ligase binding enzyme called Itch was a key player in the process. "Itch is the adapter," Kerppola says. "It tags Jun with ubiquitin, and is necessary for the protein to be targeted to the lysosome."

If Itch doesn’t recognize Jun, Kerppola explains, the level of Jun builds up in the cell, which can alter the regulation of gene transcription and cell growth. "Applying ubiquitin-mediated fluorescence technology to Jun made it possible to discover new information on how Jun turnover is controlled in cells," Kerppola says.

The technology uses complementary fragments of a fluorescent protein, which are fused to ubiquitin and to the target protein being studied. When ubiquitin is linked to the target protein, the fragments of the fluorescent protein come together and produce a bright spot of glowing color, which can be seen with a fluorescence microscope. This allows scientists to determine the location of the ubiquitinated protein in the cell.

"Location is important," Kerppola adds, "because proteins must get to their sites of action in order to do their jobs. Each protein must fulfill many different functions in different cells and in response to different stimuli. It is the variety of modifications and interactions with partners that enable the same protein to accomplish different tasks. With this technology, we are able to see the subpopulation of a protein that is modified by ubiquitin or interacts with a particular partner."

Scientists in Kerppola’s laboratory have used bimolecular fluorescence complementation methods to study protein interactions and signaling pathways, in addition to ubiquitination. While the technology should be generally applicable to most interactions, it does have some limitations. "The assay requires the two fragments of the fluorescent protein to come together," Kerppola says. "If they can’t get together, the assay doesn’t work. It’s quite good at identifying the location where something happens in the cell, but the timing of the interaction is more difficult to study, since it takes about an hour for the fluorescent proteins to become visible."

Sally Pobojewski | EurekAlert!
Further information:
http://www.umich.edu

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