TY - JOUR
T1 - The signal-anchor sequence of CYP2C1 inserts into the membrane as a hairpin structure
AU - Szczesna-Skorupa, Elzbieta
AU - Kemper, Byron
N1 - Funding Information:
This work was supported in part by a grant from the National Institutes of Health : Grant GM35897 . We thank Dr. Nikolay Tzvetkov, Department of Biophysical Chemistry, School of Medicine, Hannover, Germany, for providing the plasmid, pGEXC4S-6P-1.
PY - 2011/11/18
Y1 - 2011/11/18
N2 - Microsomal cytochrome P450s (CYPs) are anchored to the endoplasmic reticulum membrane by the N-terminal signal-anchor sequence which is predicted to insert into the membrane as a type 1 transmembrane helix with a luminally located N-terminus. We have mapped amino acids of the CYP2C1 signal-anchor, fused to Cys-free glutathione S-transferase, within the membrane by Cys-specific labeling with membrane-impermeant maleimide polyethylene glycol. At the C-terminal end of the signal-anchor, Trp-20 was mapped to the membrane-cytosol interface and Leu-19 was within the membrane. Unexpectedly, at the N-terminal end, Glu-2 and Pro-3 were mapped to the cytoplasmic side of the membrane rather than the luminal side as expected of a type 1 transmembrane helix. Similar results were observed for the N-terminal amino acids of the signal-anchor sequences of CYP3A4 and CYP2E1. These observations indicate that contrary to the current model of the signal-anchor of CYPs as a type 1 transmembrane helix, CYP2C1, CYP2E1, and CYP3A4 are monotopic membrane proteins with N-terminal signal-anchors that have a hairpin or wedge orientation in the membrane.
AB - Microsomal cytochrome P450s (CYPs) are anchored to the endoplasmic reticulum membrane by the N-terminal signal-anchor sequence which is predicted to insert into the membrane as a type 1 transmembrane helix with a luminally located N-terminus. We have mapped amino acids of the CYP2C1 signal-anchor, fused to Cys-free glutathione S-transferase, within the membrane by Cys-specific labeling with membrane-impermeant maleimide polyethylene glycol. At the C-terminal end of the signal-anchor, Trp-20 was mapped to the membrane-cytosol interface and Leu-19 was within the membrane. Unexpectedly, at the N-terminal end, Glu-2 and Pro-3 were mapped to the cytoplasmic side of the membrane rather than the luminal side as expected of a type 1 transmembrane helix. Similar results were observed for the N-terminal amino acids of the signal-anchor sequences of CYP3A4 and CYP2E1. These observations indicate that contrary to the current model of the signal-anchor of CYPs as a type 1 transmembrane helix, CYP2C1, CYP2E1, and CYP3A4 are monotopic membrane proteins with N-terminal signal-anchors that have a hairpin or wedge orientation in the membrane.
KW - Cytochrome P450
KW - Endoplasmic reticulum
KW - Membrane protein
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U2 - 10.1016/j.bbrc.2011.10.082
DO - 10.1016/j.bbrc.2011.10.082
M3 - Article
C2 - 22040732
AN - SCOPUS:84855908878
VL - 415
SP - 405
EP - 409
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -