CALNUC (nucleobindin) is localised in the Golgi apparatus in insect cells

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf 21 insect cells and selected as Golgi-specific by immunostaining of the cel ls. The antigen was purified from the cells by immunoaffinity chromatograph y 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 seque nce deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi p rotein 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, bu t had no leucine zipper which the mammalian homologues commonly have. An el ectron microscopic immunoperoxidase study demonstrated that the insect prot ein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect prot ein was considered to be a homologue of CALNUC rather than that of NEFA, As says involving proteinase K digestion, sodium carbonate extraction and Trit on X-114 extraction revealed that the insect CALNUC-like protein was a solu ble 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 th e insect protein is CALNUC, The existence of CALNUC in insect cells suggest s that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested th at NEFA was derived from CALNUC long after the segregation of a mammalian a ncestor from an insert ancestor.