Apobec-1 interacts with a 65-kDa complementing protein to edit apolipoprotein-B mRNA in vitro.
The editing of apolipoprotein-B (apoB) mRNA involves the deamination of cytidine at nucleotide 6666 to uridine. The catalytic subunit of the editing enzyme, apobec-1, is a cytidine deaminase that requires other unidentified proteins to edit apoB mRNA in vitro. We partially purified an activity from baboon kidney that functionally complements apobec-1. The complementing activity was protease-sensitive and micrococcal nuclease-resistant, had a native molecular mass of 65 +/- 10 kDa on size exclusion chromatography, and sedimented at 4.5 S in glycerol gradients. Purified recombinant His6-tagged apobec-1 immobilized on beads depleted >90% of the complementing activity from partially purified extracts. These beads edited apoB mRNA in vitro in the absence of exogenous apobec-1 or complementing activity. A functional holoenzyme containing apobec-1 and the complementing activity was eluted from the apobec-1-affinity resin using 0.5 M imidazole, whereas buffer containing 0.4 M KCl eluted only the complementing activity. The carboxyl-terminal 59 amino acids of apobec-1 were not required for interaction with the complementing activity in vitro. Our results demonstrate that the complementing protein interacts directly with apobec-1 in the absence of apoB mRNA.
Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
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Mehta
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Banerjee
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Driscoll
D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase / Q000378:metabolism*; D017353:Gene Deletion; D010215:Papio; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D001055:Apolipoproteins B; D002352:Carrier Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003562:Cytidine; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; D003564:Cytidine Deaminase