The presence of stomatin in detergent-insoluble domains of neutrophil granule membranes

Neutrophil azurophil granules, tradi- tionally regarded as the neutrophil counterpart to lysosomes, lack the lysosomal marker lysosome- associated membrane glycoprotein and have re- cently been suggested to be nonlysosomal secre- tory organelles. The membrane of the azurophil granules is poorly characterized—CD63 and CD68 are the only membrane proteins identified so far. Here, azurophil granule membranes were iso- lated by Percoll gradient subcellular fractionation. Using matrix-assisted laser desorption ionization time of flight mass spectrometry of tryptic peptides from an isolated protein, stomatin was identified in these membranes. Using immunoelectron micros- copy and immunoblot analysis of isolated or- ganelles, stomatin was found to be subcellularly localized, not only to the azurophil granules but also by a major part to the specific granules and by a minor part to the secretory vesicles/plasma mem- brane. We also show the presence of detergent- insoluble, low-density membrane domains in the plasma membrane and the granule membranes and found stomatin to be localized to these domains. J. Leukoc. Biol. 72: 970-977; 2002.