Poster abstracts

Poster number 14 submitted by Greetchen Diaz

A Possible Role of the Spindle Pole Body in Protein Targeting to the Inner Nuclear Membrane

Greetchen Diaz-Munoz (Molecular, Cellular and Developmental Biology Program, Ohio State University), Terri Harchar (Department of Biochem. and Mol. Biol. Pennsylvania State University College of Medicine, Hershey, PA), Charlie Boone (Banting & Best Department of Medical Research University of Toronto, Toronto Ontario), Anita K. Hopper (Department of Molecular Genetics, Ohio State University)

Abstract:

The overall goal of my research is to understand nuclear organization. At the nuclear envelope, the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) are connected at the nuclear pore complex (NPC) where macromolecules are exchanged between the nucleoplasm and cytosol. Nuclear membrane proteins function in gene expression and nuclear organization. How they are targeted to the INM is not well understood and most studies on INM protein targeting concern integral membrane proteins, rather than peripherally associated INM proteins. My research is focus on understanding the mechanism of protein targeting to the INM of peripherally associated proteins. I utilize Saccharomyces cerevisiae as a genetic model and Trm1, a tRNA modification enzyme as a reporter. By screening a collection of temperature sensitive (ts) mutants containing ~500 mutations of essential genes, I uncovered ~30 mutants that mislocalize galactose-inducible Trm1-GFP. At the non-permissive temperature (NPT), Trm1-GFP accumulates as a spot at the nuclear rim, rather than being evenly distributed around the entire INM. About 22% of these mutated genes encode components of the Spindle Pole Body (SPB). At NPT, the Trm1-GFP spot localizes to a region close to the ER-nucleus junction. I am currently testing the hypothesis that the spot at the ER-nucleus junction is the location where Trm1 initiates “spreading” throughout the INM and this process is dependent on functional SPBs. My observations suggest that only the INM localization of the new synthesized Trm1-GFP protein is affected by the SPB mutation and Trm1-GFP that locates to the INM before the temperature shift is not altered at NPT. In order to document the dynamics of the old and new synthesized Trm1-GFP in SPB mutants, I am currently employing different approaches that include a Trm1 double-tagged strategy and Fluorescence recovery after photobleaching (FRAP) to study Trm1-GFP dynamics.

References:
Lai T.P., Stauffer K.A., Murthi A., Shaheen H.H., Peng G., Martin N.C., Hopper A.K. 2009. Traffic

King, M. C., Lusk, P. C. and Blobel G. 2006. Nature

Murthi, A. and Hopper, A.K. 2005. Genetics

Keywords: protein targeting, inner nuclear membrane, peripheral proteins