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Conserved nuclear envelope protein uses a shuttle service to travel between job sites
Date:2/10/2014

t makes working with the gene very technically challenging. So we knew it was really important and the amount was really important, but we didn't know what it did or why it did it."

To learn more, Jaspersen and Jingjing Chen, a postdoctoral researcher in her lab, created yeast with mutations in the ndc1 gene. As expected, changes that disrupted Ndc1's interaction with its known partners in the nuclear core complex or the spindle pole body were lethal. One mutation puzzled the scientists, however. The altered Ndc1 protein bound to the expected components of both the nuclear pore complex and the spindle pole body, just like the normal protein. "Previous data suggests that if Ndc1 can functionally bind to these components, both the spindle pole body and the nuclear pore complex should be fine," Chen says. But when yeast cells produced this altered version of Ndc1, they died. "So this suggests that there may be another very critical interaction partner," she says.

Chen devised a strategy to search for such a partner. She turned to a method called a yeast two-hybrid assay, in which researchers link the proteins they are testing to two different parts of a gene activator. If the proteins associate with one another inside the cell, they bring along the gene activator components, allowing them to work together to switch on an easily detectable gene. Using a membrane-based yeast two-hybrid assay, which could detect interactions at the nuclear envelope, Chen found that Ndc1 bound to a protein called Mps3.

Jaspersen had first encountered Mps3 in 2002, when she discovered that yeast needed the protein to duplicate their spindle pole bodies prior to cell division. Mps3's interaction with Ndc1, however, was a surprise.

The institute's molecular microscopy research advisors, Brian Slaughter, Ph.D., and Jay Unruh, Ph.D., proposed that the team use a sophisticated imaging technique called fluorescence cross-correlation microscopy to look
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Contact: Gina Kirchweger
gxk@stowers.org
816-806-1036
Stowers Institute for Medical Research
Source:Eurekalert  

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