Arabidopsis dynamin-related proteins DRP2B and DRP1A participate together in clathrin-coated vesicle formation during endocytosis.
Endocytosis performs a wide range of functions in animals and plants. Clathrin-coated vesicle (CCV) formation is an initial step of endocytosis, and in animal cells is largely achieved by dynamins. However, little is known of its molecular mechanisms in plant cells. To identify dynamin-related proteins (DRPs) involved in endocytic CCV formation in plant cells, we compared the behaviors of two structurally different Arabidopsis DRPs, DRP2B and DRP1A, with those of the clathrin light chain (CLC), a marker of CCVs, at the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). DRP2B shares domain organization with animal dynamins whereas DRP1A is plant-specific. We show that green fluorescent protein (GFP)-tagged DRP2B and DRP1A colocalized with CLC tagged with monomeric Kusabira Orange (mKO) in Arabidopsis cultured cells. Time-lapse VIAFM observations suggested that both GFP-DRP2B and GFP-DRP1A appeared and accumulated on the existing mKO-CLC foci and disappeared at the same time as or immediately after the disappearance of mKO-CLC. Moreover, DRP2B and DRP1A colocalized and assembled/disassembled together at the plasma membrane in Arabidopsis cells. A yeast two-hybrid assay showed that DRP2B and DRP1A interacted with each other. An inhibitor of clathrin-mediated endocytosis, tyrphostin A23, disturbed the localization of DRP1A, but had little effect on the localization of DRP2B, indicating that DRP1A and DRP2B have different molecular properties. These results suggest that DRP2B and DRP1A participate together in endocytic CCV formation in Arabidopsis cells despite the difference of their molecular properties.
Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Masaru
M
Fujimoto
Shin-ichi
S
Arimura
Takashi
T
Ueda
Hideki
H
Takanashi
Yoshikazu
Y
Hayashi
Akihiko
A
Nakano
Nobuhiro
N
Tsutsumi
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D033941:Clathrin Light Chains / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D034281:Dynamins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020032:Tyrphostins / Q000494:pharmacology
D029681:Arabidopsis Proteins; D033941:Clathrin Light Chains; D017931:DNA Primers; D018744:DNA, Plant; D011993:Recombinant Fusion Proteins; D020032:Tyrphostins; D049452:Green Fluorescent Proteins; C539085:DRP2B protein, Arabidopsis; D019204:GTP-Binding Proteins; C475162:ADL1A protein, Arabidopsis; D034281:Dynamins; C060641:tyrphostin A23