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J Biol Chem, Vol. 274, Issue 7, 4364-4369, February 12, 1999

Biosynthesis of Flavocytochrome b₅₅₈
gp91^phox IS SYNTHESIZED AS A 65-kDa PRECURSOR (p65) IN THE ENDOPLASMIC RETICULUM

Lixin Yu, Frank R. DeLeo^¶, Karla J. Biberstine-Kinkade, Jan Renee^¶, William M. Nauseef^¶, and Mary C. Dinauer

From the  Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the ^¶ Inflammation Program and Department of Medicine, Veterans Administration Medical Center and University of Iowa, Iowa City, Iowa 52242

The redox center of the phagocyte NADPH oxidase is flavocytochrome b₅₅₈, 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) 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 stability of p65.

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