The retromer complex and clathrin define an early endosomal retrograde exit site.
Previous studies have indicated a role for clathrin, the clathrin adaptors AP1 and epsinR, and the retromer complex in retrograde sorting from early/recycling endosomes to the trans Golgi network (TGN). However, it has remained unclear whether these protein machineries function on the same or parallel pathways. We show here that clathrin and the retromer subunit Vps26 colocalize at the ultrastructural level on early/recycling endosomes containing Shiga toxin B-subunit, a well-studied retrograde transport cargo. As previously described for clathrin, we find that interfering with Vps26 expression inhibits retrograde transport of the Shiga toxin B-subunit to the TGN. Under these conditions, endosomal tubules that take the Shiga toxin B-subunit out of transferrin-containing early/recycling endosomes appear to be stabilized. This situation differs from that previously described for low-temperature incubation and clathrin-depletion conditions under which Shiga toxin B-subunit labeling was found to overlap with that of the transferrin receptor. In addition, we find that the Shiga toxin B-subunit and the transferrin receptor accumulate close to multivesicular endosomes in clathrin-depleted cells, suggesting that clathrin initiates retrograde sorting on vacuolar early endosomes, and that retromer is then required to process retrograde tubules. Our findings thus establish a role for the retromer complex in retrograde transport of the B-subunit of Shiga toxin, and strongly suggest that clathrin and retromer function in consecutive retrograde sorting steps on early endosomes.
Popoff
Vincent
V
Laboratoire Trafic et Signalisation, UMR144 Curie/CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Mardones
Gonzalo A
GA
Tenza
Danièle
D
Rojas
Raúl
R
Lamaze
Christophe
C
Bonifacino
Juan S
JS
Raposo
Graça
G
Johannes
Ludger
L
D002966:Clathrin / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022621:Shiga Toxin / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D002966:Clathrin; D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C498296:VPS26A protein, human; C419572:VPS29 protein, human; D033921:Vesicular Transport Proteins; D022621:Shiga Toxin