CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells.
A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.
Kawano
J
J
Department of Anatomy, Miyazaki Medical College, Japan.
Kotani
T
T
Ogata
Y
Y
Ohtaki
S
S
Takechi
S
S
Nakayama
T
T
Sawaguchi
A
A
Nagaike
R
R
Oinuma
T
T
Suganuma
T
T
D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D005260:Female; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D006133:Growth Substances / Q000096:biosynthesis*; D007074:Immunoglobulin G / Q000378:metabolism; D007150:Immunohistochemistry; D007313:Insecta; D008361:Mannosidases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D010802:Phylogeny; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D043323:alpha-Mannosidase
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006133:Growth Substances; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D008361:Mannosidases; D043323:alpha-Mannosidase; D000118:Acetylglucosaminidase; D002118:Calcium