Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly.
The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b(558) (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91(phox) and p22(phox) to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91(phox) precursor, gp65, was processed to gp91(phox) within 4-8 h of chase, unassembled gp65 and p22(phox) monomers were degraded by the cytosolic proteasome. gp65 associated with p22(phox) post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22(phox) coprecipitated with unglycosylated gp91(phox) primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22(phox) was unaffected. In succinyl acetone-treated cells, p22(phox) and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22(phox) heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22(phox), a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.
DeLeo
F R
FR
Inflammation Program, Department of Medicine, Veterans Affairs Medical Center and University of Iowa, Iowa City, Iowa 52242, USA.
Burritt
J B
JB
Yu
L
L
Jesaitis
A J
AJ
Dinauer
M C
MC
Nauseef
W M
WM
D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006418:Heme / Q000737:chemistry; D019255:NADPH Oxidases; D011499:Protein Processing, Post-Translational; D013211:Staphylococcus aureus
D003573:Cytochrome b Group; D006418:Heme; C036888:cytochrome b558; D019255:NADPH Oxidases