Biosynthesis of Flavocytochrome b 558

Abstract

The redox center of the phagocyte NADPH oxidase is flavocytochrome b 558 , a transmembrane protein with two subunits, gp91 phox and p22 phox . In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91 phox precursor (p65). Expressing the gp91 phox cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91 phox transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22 phox was found in subcellular compartments containing the mature 91-kDa form of gp91 phox and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91 phox and p22 phox but had little or no impact on p65. These studies demonstrate (a) gp91phox is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.

Processing and Maturation of Flavocytochromeb 558 Include Incorporation of Heme as a Prerequisite for Heterodimer Assembly

“…7, A and B). This finding supports our previous studies that suggest that the relative stabilities of gp91 phox and p22 phox are governed, in part, by the availability of heme (33,34). There was relatively more gp65 by 4 h of chase in SA-treated cells compared with that in untreated cells (Fig.…”

“…These results, in combination with the data presented above, demonstrate that the formation of flavocytochrome b heterodimers required neither N-linked glycosylation nor transit into the Golgi complex. Heme Acquisition and Heterodimer Formation-The binding of heme by flavocytochrome b has been implicated in the stability of gp91 phox and p22 phox (33,34). To determine whether the binding of heme was necessary for the maturation of flavocytochrome b, gp91-PLB cells were cultured in the presence of SA, an inhibitor of 5-aminolevulinic dehydratase and thus heme synthesis (Fig.…”

“…phox is synthesized as a 65-kDa precursor in the ER (34). Cells were chased with unlabeled methionine to follow the processing of gp65 to mature gp91 phox and to elucidate the time point during synthesis at which it associated with p22 phox .…”

Intracellular Localization and Preassembly of the NADPH Oxidase Complex in Cultured Endothelial Cells

“…In neutrophil membrane preparations, gp91 phox typically migrates as multiple bands or a smear between ϳ65 and ϳ100 kDa on SDS-PAGE because of variable post-translational protein glycosylation (37)(38)(39). In the present study, two different anti-gp91 phox antibodies both detected bands between ϳ75 and ϳ90 kDa in endothelial cell protein as well as neutrophil membrane, suggesting that authentic gp91 phox with variable glycosylation was being detected.…”

Mutagenesis of p22 Histidine 94

“…Co-expression of gp91 phox and wild type p22 phox has been shown to increase the expression of gp91 phox in PLB-985 and Cos7 cells and to increase maturation of gp91 phox from the 65-kDa precursor to the fully glycosylated ϳ 91-kDa form (33). As in the p22 phox -transfected Cos7 cells, Western blot analysis showed that protein levels of the p22 phox mutants H94Y (Fig.…”