Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes

In this study we have used the Semliki forest virus expression system to tr ansiently express chimeric proteins that contain transmembrane and cytoplas mic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studie s showed that the chimeric protein with the entire cytoplasmic domain of CI -MPR was transported to late endosomes, where it accumulated. We made use o f the biotin-binding capacity of lumenal avidin, and found that, in agreeme nt with this distribution, the chimeric protein could be labelled with biot inylated HRP endocytosed for a long, but not a brief, period of time. Howev er, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulat ed within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or f or longer periods at 19 degrees C). Coinfection of these chimeras showed th at they follow the same route from the TGN to the early endosomes. We concl ude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results a lso suggest that the YKYSKV sequence close to the CI-MPR transmembrane segm ent is sufficient for targeting to sorting early endosomes.