Characterization of the in vitro retrograde transport of MPR46.
The mannose 6-phosphate receptor MPR46 mediates sorting of lysosomal enzymes and recycles between the trans-Golgi network and endosomes. We characterized the retrograde transport of MPR46 from endosomes to the TGN by an in vitro transport assay using mouse fibroblast cell lines. Sulfation of a modified MPR46 upon entering the TGN is measured. The in vitro retrograde transport is time-, temperature-, ATP- and cytosol-dependent. Transport requires the SNARE proteins Vti1a and Syntaxin 16 and the Rab family member Rab6. The transport is sensitive to GTP gamma S, brefeldin A and independent of TIP47. These data indicate that MPR46 follows an early endosome-to-TGN route. Transport is inhibited by MPR46 tail peptide comprising the acidic cluster-di-leucine sorting motif to which adaptor proteins AP-1 and AP-3 bind. Transport depends on cytosolic AP-3, but not on cytosolic AP-1. Residual membrane-associated AP-1 may have masked a requirement for cytosolic AP-1. The competence of membranes from AP-1-deficient cells for endosome-to-TGN transport in vitro was severely compromised.
Medigeshi
Guruprasad Ramarao
GR
Georg-August Universität Göttingen, Zentrum für Biochemie und Molekulare Zellbiologie, Biochemie II, Heinrich-Düker-Weg-12, D-37073 Goettingen, Germany.
Schu
Peter
P
D033961:Adaptor Protein Complex 1 / Q000378:metabolism; D033963:Adaptor Protein Complex 3 / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology*; D015415:Biomarkers; D020126:Brefeldin A / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D010455:Peptides / Q000378:metabolism; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D021381:Protein Transport; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D033921:Vesicular Transport Proteins; D021601:trans-Golgi Network / Q000378:metabolism*
D033961:Adaptor Protein Complex 1; D033963:Adaptor Protein Complex 3; D015415:Biomarkers; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C038196:PLIN3 protein, human; D010455:Peptides; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D011500:Protein Synthesis Inhibitors; D017527:Receptor, IGF Type 2; D033921:Vesicular Transport Proteins; D020126:Brefeldin A; D000255:Adenosine Triphosphate