title: The membrane-interactive tail of cytochrome b(5) can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum.
abstract: Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b(5) indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins. Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences. A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b(5), including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b(5) in the endoplasmic reticulum membrane. Precytochrome b(5) was cotranslationally translocated across the endoplasmic reticulum membrane. The signal-processed cytochrome b(5) was integrally anchored in the membrane with the globular domain facing the lumen. Thus, the topology of the signal sequence-directed cytochrome b(5) in the microsomal vesicle was reversed with respect to that of the native form. Posttranslational incubation of the precytochrome b(5) with microsomes resulted in a "loose" incorporation of the unprocessed form onto the surface of the vesicle. Our findings suggest that the membrane-insertion sequence of cytochrome b(5) has a functional stop-transfer sequence. We discuss the implications of these findings with respect to selective targeting of cytochrome b(5) to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.
authors.affiliation: Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK. mak@aber.ac.uk
authors.forename: Mustak A
authors.initials: MA
authors.lastname: Kaderbhai
authors.forename: Rachel
authors.initials: R
authors.lastname: Morgan
authors.forename: Naheed N
authors.initials: NN
authors.lastname: Kaderbhai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D015394:Molecular Structure; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction
chemical_list: D008565:Membrane Proteins; D021382:Protein Sorting Signals; D004247:DNA; D015786:Cytochromes b5