title: Methods of analysis of the membrane trafficking pathway from recycling endosomes to lysosomes.
abstract: The transferrin receptor (TfR) is responsible for iron uptake through its trafficking between the plasma membrane and recycling endosomes, and as a result it has become a well-known marker for recycling endosomes. Although the molecular basis of the TfR recycling pathway has been thoroughly investigated, the TfR degradation mechanism has been poorly understood. Exposure of cultured cells to two drugs, the protein synthesis inhibitor cycloheximide and the V-ATPase inhibitor bafilomycin A1, recently showed that TfR is not only recycled back to the plasma membrane after endocytosis but is constitutively transported to lysosomes for degradation. The results of genome-wide screening of mouse Rab small GTPases (common regulators of membrane trafficking in all eukaryotes) have indicated that Rab12 regulates TfR trafficking to lysosomes independently of the known membrane trafficking pathways, for example, the conventional endocytic pathway and recycling pathway. This chapter summarizes the methods that the authors used to analyze the membrane trafficking pathway from recycling endosomes to lysosomes that is specifically regulated by Rab12.
authors.affiliation: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Takahide
authors.initials: T
authors.lastname: Matsui
authors.affiliation: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan. Electronic address: nori@m.tohoku.ac.jp.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukuda
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D066246:ErbB Receptors; D049452:Green Fluorescent Proteins; D051907:Lysosomal Membrane Proteins; D018942:Macrolides; D020033:Protein Isoforms; D011990:Receptors, Transferrin; D020691:rab GTP-Binding Proteins; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1; C485184:enhanced green fluorescent protein; C040929:bafilomycin A1; C541485:EGFR protein, mouse; C569728:rab12 protein, mouse
keywords: Degradation; Lysosome; Membrane traffic; Rab12; Recycling endosome; Small GTPase; Transferrin receptor