Genome wide screen reveals a new role for acetylation in nuclear membrane targeting of proteins.
Athulaprabha Murthi, Anita. K. Hopper
Dept of Biochem & Mol Biol, Penn State University, 500 Univ Dr, Hershey, PA, 17033, U.S.A
The nucleus is surrounded by a double-layered membrane, an inner nuclear membrane (INM) and an outer nuclear membrane (ONM). Many integral membrane proteins span the lipid bilayer while the peripheral proteins are tethered to the membrane via protein-protein, or protein-lipid interactions. We conducted a genome-wide screen to understand how proteins are targeted to the INM. We employed a GFP tagged protein Trm1p-GFP as a reporter. Trm1p is a tRNA methyltransferase that is peripherally associated with the INM. We reasoned that deletions of genes important for targeting or tethering proteins to the INM would cause mislocalization of the Trm1p-GFP. Strains of the deletion collection were transformed, with a plasmid encoding Trm1p-GFP, in the 96 well format and assayed visually for its sub nuclear localization. To date, ~4000 strains have been screened. Of these, four cause Trm1p-GFP to be nucleoplasmic. Three contain deletions of genes encoding subunits of a N-terminal acetyltransferase complex, Nat C. Mutations at the N-terminus of Trm1p-GFP also cause it to be nucleoplasmic, leading to the model that Trm1p is a Nat C substrate. However, we have not eliminated the possibility that Nat C affects Trm1p-GFP localization indirectly. We are in the process of distinguishing between these two possibilities. We also uncovered a gene encoding a putative integral membrane protein that may serve as an INM tether for peripheral proteins or function in nuclear membrane structure.
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