Biosynthesis of flavocytochrome b(558) - gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum

The redox center of the phagocyte NADPH oxidase is flavocytochrome b(558), a transmembrane protein with two subunits, gp91(phox) and p22(phox). In thi s study we investigated the identity, subcellular localization, and maturat ion of a putative 65-kDa gp91(phox) precursor (p65), Expressing the gp91(ph ox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes, Sucrose d ensity gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91(phox) transg ene demonstrated that p65 co-sedimented with the ER marker protein calretic ulin 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 occ ur in a post-ER compartment, The heme synthesis inhibitor, succinyl acetone , reduced the abundance of mature gp91(phox) and p22(phox) but had little o r no impact on p65, These studies demonstrate (a) gp91(phox) 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 st ability of p65.