title: Cytochrome b(5) forms homomeric complexes in living cells.
abstract: Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.
authors.affiliation: Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600, South Africa.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Storbeck
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Swart
authors.forename: Nicolaas
authors.initials: N
authors.lastname: Lombard
authors.forename: Craig V
authors.initials: CV
authors.lastname: Adriaanse
authors.forename: Pieter
authors.initials: P
authors.lastname: Swart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D034561:Sheep, Domestic; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism
chemical_list: D015786:Cytochromes b5; D013254:Steroid 17-alpha-Hydroxylase