Retention of cytochrome b5 in the endoplasmic reticulum is transmembrane and luminal domain-dependent.
Cytochrome b5 (b5), a typical tail-anchored protein of the endoplasmic reticulum (ER) membrane, is composed of three functionally different domains: amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxyl-terminal ER-targeting domains (Mitoma, J., and Ito, A. (1992) EMBO J. 11, 4197-4203). To analyze the potential retention signal of b5, mutant proteins were prepared to replace each domain with natural or artificial sequences, and subcellular localizations were examined using immunofluorescence microscopy and cell fractionation. The transmembrane domain functioned to retain the cytochrome in the ER, and the mutation of all or part of the transmembrane domain with an artificial hydrophobic sequence had practically no effect on intracellular distribution of the cytochrome. However, when the transmembrane domain was extended systematically, a substantial portion of the protein with the domain of over 22 amino acid residues leaked from the organelle. Thus, the transmembrane length functions as the retention signal. When cytochromes with mutations at the carboxyl-terminal end were overexpressed in cells, a substantial portion of the protein was transported to the plasma membrane, indicating that the carboxyl-terminal luminal domain also has a role in retention of b5 in the ER. Carbohydrate moiety of the glycosylatably-mutated b5 was sensitive to endoglycosidase H but resistant to endoglycosidase D. Therefore, both transmembrane and carboxyl-terminal portions seems to function as the static retention signal.
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
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M
Honsho
J Y
JY
Mitoma
A
A
Ito
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D015786:Cytochromes b5 / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015398:Signal Transduction
D015786:Cytochromes b5