TARGETING OF PROTEINS TO GRANULE SUBSETS IS DETERMINED BY TIMING AND NOT BY SORTING - THE SPECIFIC GRANULE PROTEIN NGAL IS LOCALIZED TO AZUROPHIL GRANULES WHEN EXPRESSED IN HL-60 CELLS

The mechanism of protein targeting to individual granules in cells tha t contain different subsets of storage granules is poorly understood, The neutrophil contains two highly distinct major types of granules, t he peroxidase positive (azurophil) granules and the peroxidase negativ e (specific and gelatinase) granules. We hypothesized that targeting o f proteins to individual granule subsets may be determined by the stag e of maturation of the cell, at which the granule proteins are synthes ized, rather than by individual sorting information present in the pro teins, This was tested by transfecting the cDNA of the specific granul e protein, NGAL, which is normally synthesized in metamyelocytes, into the promyelocytic cell line HL-60, which is developmentally arrested at the stage of formation of azurophil granules, and thus does not con tain specific and gelatinase granules. Controlled by a cytomegalovirus promoter, NGAL was constitutively expressed in transfected HL-60 cell s, This resulted in the targeting of NGAL to azurophil granules as dem onstrated by colocalization of NGAL with myeloperoxidase, visualized b y immunoelectron microscopy. This shows that targeting of proteins int o distinct granule subsets may be determined solely by the time of the ir biosynthesis and does not depend on individual sorting information present in the proteins.