cDNA sequence and bacterial expression of mouse liver sterol carrier protein-2.
Sterol carrier protein-2 (SCP-2) is an intracellular protein of Mr 13,096. In vitro studies have shown that it is involved in the transport and metabolism of cholesterol. This protein is believed to participate in these activities by forming a stoichiometric complex with the sterol. Because these activities occur in different intracellular locations, i.e. mitochondria, peroxisomes, and cytosol, it can be predicted that SCP-2 targets to these sites. In this report we show that a mouse cDNA (785 base pairs) encodes a precursor form of SCP-2 containing a N-terminal presequence and an additional C-terminal residue. These additional amino acid residues are found in proteins targeted to the mitochondria and peroxisomes, respectively. These signals are not found in SCP-2 purified from rat liver cytosol which is believed to be a cytosolic form. Northern analysis shows that there are four species of mRNA which hybridize to a SCP-2-specific probe at 1.0, 1.7, 2.2, and 2.9 kilobases. Southern analysis shows that the gene is distributed over a large amount of DNA or that there are multiple genes. We have cloned the cytosolic/peroxisomal form of mouse SCP-2 into the Escherichia coli expression vector pKK233-2 and have expressed and purified recombinant mouse SCP-2, Mr 13,034. The purified recombinant SCP-2 is immunoreactive to rabbit anti-rat SCP-2 antibody. It also has biological activity equivalent to homogeneous rat liver SCP-2 in stimulating the microsomal conversion of 7-dehydrocholesterol to cholesterol and in the esterification of cholesterol by acyl-CoA cholesterol acyltransferase by rat liver microsomes.
Moncecchi
D
D
Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque 87131.
Pastuszyn
A
A
Scallen
T J
TJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D002788:Cholesterol Esters / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010940:Plant Proteins; D010957:Plasmids; D012313:RNA / Q000032:analysis; D051381:Rats; D015183:Restriction Mapping; D013261:Sterols / Q000378:metabolism*
D000596:Amino Acids; D002352:Carrier Proteins; D002788:Cholesterol Esters; D010940:Plant Proteins; D013261:Sterols; C072087:sterol carrier proteins; D012313:RNA; D004247:DNA