Holocarboxylase synthetase (HCS) catalyzes the biotinylation of three mitochondrial and one cytosolic forms of biotin-dependent carboxylases in humans. Patients suffering from this autosomal recessive disease have Multiple Carboxylase Deficiency (MCD) with symptoms of life-threatening metabolic acidosis which, in almost all cases, can be successfully treated with pharmacologic doses of oral biotin. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS maybe targeted to the cytoplasm and mitochondria or that carboxylases are biotinylated in the cytoplasm prior to import into the mitochondria. In order to resolve the compartmentalization of HCS, 5' HCS cDNA sequences have been examined for a targeting signal and a candidate sequence was tested for its capacity to target mitochondria. Analysis of 5' cDNA reveals complex alternative splicing, none of which appear to contain mitochondrial targeting sequences. In addition, antibodies have been developed in order to perform immunochemical analysis of the subcellular distribution of HCS. Polyclonal antisera were raised against full length HCS as well as two peptides corresponding to a 20 amino acid region in the N-terminus and to the 20 amino acids preceding the stop codon. Immunohistochemical staining of human fibroblasts with the antibody to full length HCS gives cytosolic, mitochondrial and nuclear localization. Interestingly, analysis with the N-terminal antiserum reveals a large punctate staining pattern exclusively localized to the nucleus. The corresponding C-terminal antiserum reveals solid nuclear staining with some mitochondrial co-localization. Taken together, these results indicate the ubiquitous nature of HCS in human cells and also allude to a potential role for HCS in the nucleus of human cells.

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