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

Abstract

Neutrophil azurophil granules, traditionally regarded as the neutrophil counterpart to lysosomes, lack the lysosomal marker lysosome-associated membrane glycoprotein and have recently been suggested to be nonlysosomal secretory 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 isolated 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 microscopy and immunoblot analysis of isolated organelles, 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 membrane. 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.