SUBCELLULAR-DISTRIBUTION OF DOCKING FUSION PROTEINS IN NEUTROPHILS, SECRETORY-CELLS WITH MULTIPLE EXOCYTIC COMPARTMENTS/

Neutrophils contain at least four distinct types of secretory organell es, which undergo exocytosis during infection and inflammation, The si gnaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional di fferences in docking and fusion with the plasma membrane, As a step to ward understanding the processes underlying differential granular secr etion in neutrophils, we assessed the presence and distribution of a n umber of proteins reported to be involved in vesicular docking and/or fusion in other systems, Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron micr oscopy, Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, syna ptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevi n were not detectable in human neutrophils. In contrast, syntaxin 4, V AMP-2, and the 39-kDa isoform of secretory carrier membrane protein (S CAMP) were present, SCAMP was found mainly in secondary and tertiary g ranules and in a fraction containing secretory vesicles, but was virtu ally absent from the primary (lysosomal) granules, This profile is con sistent with the proposed ''post-Golgi'' distribution of SCAMP, VAMP-2 was largely absent from primary and secondary granules, but concentra ted in tertiary granules and secretory vesicles. This pattern of distr ibution parallels the increasing sensitivity of these exocytic compart ments to intracellular free calcium. Accordingly, ionomycin induced tr anslocation of VAMP-P toward the plasma membrane, Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lame llipodia of migrating cells, This regional accumulation may contribute to localized secretion into the phagosomal lumen.