RETROGRADE LIPID TRAFFIC IN YEAST - IDENTIFICATION OF 2 DISTINCT PATHWAYS FOR INTERNALIZATION OF FLUORESCENT-LABELED PHOSPHATIDYLCHOLINE FROM THE PLASMA-MEMBRANE

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Tw o pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature ( <2-degrees-C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mu tations in the SEC63, SEC65, or SEC11 genes, required for translocatio n of nascent secretory polypeptides into the ER or signal peptide proc essing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin he avy chain, CHC1. The correlation of biochemical analysis with fluoresc ence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, d ependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced ) and was largely independent of the SEC genes. Non-endocytic transpor t of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N- ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of p hosphatidylcholine across the yeast plasma membrane. These data establ ish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracell ular organelles through non-endocytic, protein-mediated events.