The cytoplasmic and N-terminal transmembrane domains of cytochrome P450 contain independent signals for retention in the endoplasmic reticulum.
Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.
Szczesna-Skorupa
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E
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign 61801, USA.
Ahn
K
K
Chen
C D
CD
Doray
B
B
Kemper
B
B
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; C084930:cytochrome P-450 2C2; C096409:CYP2C1 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D066246:ErbB Receptors; D000469:Alkaline Phosphatase