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Subcellular distribution of the Rap1A protein in human neutrophils:
colocalization and cotranslocation with cytochrome b559
MT Quinn, ML Mullen, AJ Jesaitis and JG Linner
Department of Chemistry and Biochemistry, Montana State University, Bozeman
59717.
Rap1A, a low molecular weight guanosine triphosphate-binding protein
(LMWG), has been shown previously by us to be associated with purified
cytochrome b from stimulated human neutrophils. In the present studies, we
show that Rap1A is also associated with affinity-purified cytochrome b from
unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to
biochemically and immunocytochemically determine the subcellular
distribution of Rap1A in resting and activated human neutrophils. Analysis
of the subcellular fractionation of unstimulated cells by Western blotting
of isopycnic sucrose density gradient fractions with anti-Rap1 peptide
antibodies indicated that Rap1A colocalized with cytochrome b in the plasma
membrane as well as in the specific granule membranes and that it was
translocated, along with cytochrome b, to the plasma membrane when the
cells were stimulated with phorbol myristate acetate (PMA). No evidence for
a cytosolic localization of Rap1A was found in our studies; however, if the
cells were disrupted by sonication, rather than N2 cavitation, a fraction
of the Rap1A was released from the membrane. Electron microscopy of thin
sections of cryofixed, molecular-distillation dried neutrophils labeled
with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti-
cytochrome b peptide antibodies confirmed our biochemical localization, and
quantitation showed that more than half of the specific granule- associated
Rap1A was translocated to the plasma membrane in PMA- stimulated cells.
Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus
also demonstrated the translocation of Rap1A with cytochrome b.
Approximately 70% of the total Rap1A labeling was associated with the
phagolysosomal membrane, the site of assembly of the superoxide-generating
system. The colocalization and cotranslocation of Rap1A with cytochrome b
in resting and activated neutrophils is consistent with a functional
association of these two molecules in the intact cell and provides further
evidence for a role of this LMWG in the structure or function of the
neutrophil superoxide- generating system.
Volume 79,
Issue 6,
pp. 1563-1573,
03/15/1992
Copyright © 1992 by The American Society of Hematology
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