Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation

Abstract

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with SpG membrane proteins and a 48,000-dalton protein that was also a major component of AzG membranes. Also, four iodinated cell surface proteins co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface.Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore-induced exocytosis revealed that there was not a dramatic qualitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of 1251 that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudate PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.Numerous morphological studies have demonstrated that in secretory cells membrane-bounded granules fuse with the plasma membrane during secretion (exocytosis), resulting in the mixing of these membranous compartments (t). In addition, recent evidence suggests that components of secretory cell plasma membranes may recycle back to the Golgi complex to be reutilized for secretory granule formation (2, 3). Moreover, there are indications that in some cells newly added secretory granule membranes may recycle, intact, from the plasma membrane back to the cytoplasm (4). Even with this information, the compositional relationship between the plasmalemma and secretory granule membranes is not well documented, owing to the difficulty in obtaining plasma and granule membranes from the same cell type. Rabbit polymorphonuclear heterophils (PMNs), since they exist as single cells in vivo, possess a readily accessible plasma membrane, two well-defined populations of cytoplasmic granules, and a capacity for secreting the contents of these granules into the extraceUular space. Thus, these cells provide an excellent model for studying the relationship between the plasma membrane and secretory granule membranes.In the preceding paper (5) we reported on the separation of rabb...

Selective internalization of granule membrane after secretion in mast cells.

“…The appearance on the cell surface of additional molecular species, characteristic of secretory granule membrane, has previously been reported for other cells (ref. 25 …”

The Lysophospholipid Acyltransferase Antagonist CI‐976 Inhibits a Late Step in COPII Vesicle Budding

“…For thin‐section, transmission electron microscopy, cells were fixed in a combination of glutaraldehyde/osmium tetroxide fixative and processed as described (57,58). Images were captured on an FEI/Philips Morgagni transmission electron microscope using a Hamamatsu digital camera controlled by software from Advanced Microscopy Techniques, Corp. All images were taken at a magnification of 22 000, and from these digital images, vesicle and budding vesicle profiles at ERES were quantified at a working magnification of 44 000.…”

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

“…In the present report, the membrane and content proteins of SpG and AzG from rabbit PMNs have been characterized by ultrastrucrural, electrophoretic and radiolabeling methods. In the accompanying report we examine the radiolabeling pattern of the cell surface of rabbit PMNs before and after secretion (5). A preliminary account of this work appeared earlier (6).…”