The amino-terminal structures that determine topological orientation of cytochrome P-450 in microsomal membrane.
We previously showed that the amino-terminal region of P-450 is responsible not only for targeting to endoplasmic reticulum (ER) membrane but also for stable anchoring to the membrane. In the present study, we introduced several mutations or deletions into the signal-anchor region of the chimeric proteins in which the amino-terminal regions of two forms of cytochrome P-450 were fused to the mature portion of interleukin 2. The amino-terminal acidic amino acid residues were replaced with basic amino acid residues or the hydrophobic core sequences were partially deleted, and these mutant proteins were assayed in vitro for their capacity to be inserted into or translocated across the ER membrane. The proteins that received the former manipulations were processed and the IL-2 portion was translocated across the membrane. In one case, the processing did not occur, thereby enabling the chimeric protein to anchor on the luminal side of the ER. Those that received the latter manipulation were also processed and the IL-2 portion translocated across the ER. These results strongly suggest that the signal-anchor function is determined both by the amino-terminal charged amino acid residues and by the length of the hydrophobic stretch.
Sato
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Sakaguchi
M
M
Mihara
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum; D005033:Ethylmaleimide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D005033:Ethylmaleimide