The amino-terminal 29 amino acids of cytochrome P450 2C1 are sufficient for retention in the endoplasmic reticulum.
Cytochromes P450 are inserted into and anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic signal sequence at the NH2 terminus. To determine whether the NH2-terminal sequence might also have an ER retention function, the NH2-terminal 29 amino acids of cytochrome P450 2C1, with and without an additional 29 amino acids containing an N-glycosylation site, were fused either to a soluble cytoplasmic protein, Escherichia coli beta-galactosidase, or to a secreted protein, E. coli alkaline phosphatase, and the hybrid proteins were expressed in COS1 cells. Subcellular fractionation indicated that both the beta-galactosidase and alkaline phosphatase hybrid proteins cosedimented with marker enzymes for ER membranes, and localization by immunofluorescent staining was consistent with an ER location. Hybrid proteins with the NH2-terminal glycosylation site were glycosylated in COS1 cells, and the carbohydrate moiety was sensitive to endoglycosidase H digestion, providing further evidence that the proteins were retained in the ER. In vitro studies of membrane insertion of the alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase did not alter the topological properties of the cytochrome P450 NH2-terminal sequence. In addition, alkaline phosphatase fused to the extracellular and transmembrane domains of epidermal growth factor receptor was transported to the plasma membrane in COS1 cells, which establishes that alkaline phosphatase as a cytoplasmic domain does not prevent transport from the ER. These observations indicate that the large cytoplasmic domain of cytochrome P450 is not required for retention in the ER and suggest that a specific sequence or structure within the NH2-terminal 29 amino acids functions as an ER retention signal.
Ahn
K
K
Department of Physiology and Biophysics, University of Illinois, Urbana-Champaign, 61801.
Szczesna-Skorupa
E
E
Kemper
B
B
D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D000596:Amino Acids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D066246:ErbB Receptors; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase