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The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.

The annexins are Ca(2+)-regulated, phospholipid-binding proteins which have been suggested to take part in cellular events such as exocytosis. The subcellular localization of annexins in human neutrophils was determined using monoclonal antibodies against annexins I, II, IV and VI and a polyclonal peptide antiserum against an annexin consensus sequence. Several annexins were translocated to the light membrane fraction enriched in plasma membranes and secretory vesicles. Annexins were associated also with the azurophil and specific granules. Whereas annexins I, IV and VI and one unidentified 35 kDa protein translocated to each of the isolated organelles, annexin II, a 66 kDa annexin IV-like protein, and a 38 kDa annexin I-like protein exhibited organelle-related differences in their association with membranes. The 38 kDa annexin associated only with specific granules and the secretory vesicles/plasma membrane but not with azurophil granules. Annexin II and the 66 kDa annexin IV-like protein associated with each of the neutrophil organelles, but the binding to specific granules and secretory vesicles/plasma membrane showed a Ca(2+)-dependency different from that of azurophil granules. This observation suggests that these proteins may contribute to the secretory process in neutrophils.