Nesselhut J, Jurgan U, Onken E, Götz H, Barnikol H U, Hirschfeld G, Barnikol-Watanabe S, Hilschmann N
Department of Immunochemistry, Max-Planck-Institute for Experimental Medicine, Hermann-Rein Str. 3, D-37075 Göttingen, Germany.
FEBS Lett. 2001 Dec 14;509(3):469-75. doi: 10.1016/s0014-5793(01)03187-8.
The subcellular localization of the human Ca(2+)-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the DeltaN mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif.
