Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region.
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.
Nesselhut
J
J
Department of Immunochemistry, Max-Planck-Institute for Experimental Medicine, Hermann-Rein Str. 3, D-37075 Göttingen, Germany.
Jurgan
U
U
Onken
E
E
Götz
H
H
Barnikol
H U
HU
Hirschfeld
G
G
Barnikol-Watanabe
S
S
Hilschmann
N
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D002455:Cell Division / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C000630650:NUCB2 protein, human; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D049452:Green Fluorescent Proteins; D003513:Cycloheximide; D007930:Leucine