GTPrS-dependent phospholipase D activity in human neutrophils was investigated using exogenous phospholipid as a substrate. Both cytosolic and membrane- associated phospholipase D activities were identified. The previously described 50 kDa cytosolic activating factor was resolved chromatographically from the cytosolic phospholipase D. Using exogenous phospholipid as substrate along with chromatographically resolved 50 kDa factor and recombinant ADP-ribosylation factor 1, plasma membrane was required for activity, indicating that the activity which was previously seen using endogenous phospholipid substrate was due to a phospholipase D located in the plasma membrane. In addition, ADP-ribosylation factor and the 50 kDa factor activated synergistically. Using neutrophil plasma membranes, a third regulator of neutrophil membrane phospholipase D was identified from bovine brain cytosol. This factor was resolved from ADP-ribosylation factor and Rho A by successive column chromatographies. The brain factor showed a synergistic effect with the 50 kDa neutrophil activator but an additive effect with recombinant ADP- ribosylation factor. Whether or not ADP-ribosylation factor or the brain factor were present, high activities were seen only when the 50 kDa factor was present, indicating that the 50 kDa cytosolic factor is a major activating factor for the neutrophil plasma membrane phospholipase D.
ADP-Ribosylation Factor 1 ; ADP-Ribosylation Factors* ; Brain* ; Cell Membrane ; Chromatography ; Cytosol* ; Fibrinogen ; Humans ; Membranes ; Neutrophils* ; Phospholipase D* ; Phospholipases*

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
ADP-Ribosylation Factor 1 ; chemistry ; metabolism ; Animals ; Coatomer Protein ; chemistry ; metabolism ; Cytosol ; chemistry ; metabolism ; GTPase-Activating Proteins ; chemistry ; metabolism ; Humans ; Membranes, Artificial ; Rats