Vps10p cycles between the TGN and the late endosome via the plasma membrane in clathrin mutants.
Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in a chc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport in chc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, in chc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normal VPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.
Deloche
Olivier
O
Howard Hughes Medical Institute, University of California, Berkeley, California 94720-3206, USA.
Schekman
Randy W
RW
D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005838:Genotype; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013347:Subcellular Fractions; D013997:Time Factors; D033921:Vesicular Transport Proteins; D021601:trans-Golgi Network / Q000378:metabolism*
D002966:Clathrin; D008164:Luminescent Proteins; C087335:PEP1 protein, S cerevisiae; D011956:Receptors, Cell Surface; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D004247:DNA