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 transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated 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 for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.
Juuti-Uusitalo
K
K
University of Jyväskylä, Department of Biological and Environmental Science, Finland.
Airenne
K J
KJ
Laukkanen
A
A
Punnonen
E L
EL
Olkkonen
V M
VM
Gruenberg
J
J
Kulomaa
M
M
Marjomäki
V
V
D020816:Amino Acid Motifs; D000818:Animals; D001360:Avidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D019921:Biotinylation; D020126:Brefeldin A / Q000494:pharmacology; D002412:Cations; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011205:Povidone / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics; D012822:Silicon Dioxide / Q000494:pharmacology; D013997:Time Factors
D002412:Cations; D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D001360:Avidin; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone