A NONEXCHANGEABLE FLUORESCENT PHOSPHOLIPID ANALOG AS A MEMBRANE TRAFFIC MARKER OF THE ENDOCYTIC PATHWAY

The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was inserted into the plasma membrane of Baby hamster kidney cells at low temperature (2 degrees C). The mobility characteristics of the analog--as revealed by fluorescence photobleaching recovery--were very similar to those of membrane-inserted 1-acyl-2[6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]caproyl] phosphatidylcholine (C6-NBD-PC). Upon warming to 37 degrees C, followed by a 1-h incubation, all N-Rh-PE was located intracellularly. By contrast, after the same time interval, approximately 10% of the cell-associated PC-derivative was found intracellularly. Furthermore, the analogs moved to different intracellular sites, as N-Rh-PE associates with perinuclear and peri-Golgi structures, whereas C6-NBD-PC appears mainly in the Golgi complex. Colocalization with organelle-specific probes and Percoll gradient analysis identified the N-Rh-PE-labeled structures as lysosomes. Temperature and energy-dependent experiments supported the endocytic pathway as the mechanism of N-Rh-PE internalization. The mechanism of N-Rh-PE internalization appears to differ from that of C6-NBD-PC. In conjunction with a difference in the efficiency of removal of the lipid derivatives from the plasma membrane, the results suggest that N-Rh-PE is selectively internalized, implying that sorting of the lipid analogs already occurs at the level of the plasma membrane. The distinct difference in physical appearance of the probes after membrane insertion, i.e., N-Rh-PE being present as small clusters and C6-NBD-PC as monomers, could explain the selective sorting and internalization of N-Rh-PE. The results demonstrate that N-Rh-PE may serve as a useful marker for studying membrane traffic during endocytosis

INTERACTION OF CLATHRIN WITH LARGE UNILAMELLAR PHOSPHOLIPID-VESICLES AT NEUTRAL PH - LIPID DEPENDENCE AND PROTEIN PENETRATION

The interaction of clathrin with large unilamellar vesicles of various lipid compositions has been examined at neutral pH. Clathrin induces leakage of contents of vesicles that contain the acidic phospholipid phosphatidylserine. Leakage is greatly enhanced by the presence of a relatively minor amount of cholesterol, but is inhibited by phosphatidylcholine. Resonance energy transfer measurements between tryptophan residues of the protein and a fluorescent lipid analog, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine incorporated into the liposomal bilayer, suggests a dynamic interaction of clathrin with the bilayer at neutral pH. This interaction includes a (partial) penetration of the protein into the lipid bilayer, as revealed by hydrophobic photoaffinity labeling with 3-(trifluoromethyl)-3-(m-[I-125]iodophenyl)-diazirine. The interaction of clathrin with lipid vesicles at neutral pH is inhibited when the protein is pretreated with trypsin or with the reducing agent dithiothreitol, suggesting that structural requirements govern clathrin-membrane interaction at these conditions. The physiological relevance of the present observations in light of vesiculation and endosomal maturation is discussed