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.
phox H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91 phox H119L or H210L. In contrast, gp65 was not processed to gp91 phox , heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91 phox
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