Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin.
Clathrin-mediated endocytosis is a well-studied uptake mechanism for nutrients and signalling receptors in mammalian cells that depends on the coordinated interaction of coat proteins and endocytic network proteins to perform the internalization. In this process AP180 promotes the assembly of clathrin triskelia into coated membrane patches at the plasma membrane, while alpha-adaptin interacts with various network proteins that are in turn required for the budding of the coated pits. The process of clathrin-mediated endocytosis in plants has not been dissected at the molecular level, nor have the members of an analogous uptake machinery been functionally described. In this respect, we have investigated the AP180 and alpha-adaptin orthologs from Arabidopsis thaliana: At-AP180 and At-alphaC-Ad. Both plant proteins display the same structural features as their mammalian counterparts and fulfill the same basic functions. To identify their interacting partners, the ear region of At-alphaC-Ad and the C-terminal region of At-AP180 were used as fusion proteins in pull-down experiments and plasmon-resonance measurements. At-alphaC-Ad binds several mammalian endocytic proteins, and its interaction with At-AP180 requires the DPF motif. At-AP180 functions as a clathrin assembly protein that promotes the formation of cages with an almost uniform size distribution. Deletion of the single DLL motif abolished the assembly activity of At-AP180 almost completely, but did not affect its binding to triskelia, suggesting the existence of additional binding determinants.
Barth
Meike
M
University of Heidelberg, Department of Cell Biology, Heidelberg Institute for Plant Sciences, 69120 Heidelberg, Im Neuenheimer Feld 230, Germany.
Holstein
Susanne E H
SE
D033965:Adaptor Protein Complex alpha Subunits / Q000737:chemistry*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D001672:Biocompatible Materials; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002966:Clathrin / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008957:Models, Genetic; D008969:Molecular Sequence Data; D034041:Monomeric Clathrin Assembly Proteins / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
D033965:Adaptor Protein Complex alpha Subunits; D001672:Biocompatible Materials; D002966:Clathrin; D017931:DNA Primers; D034041:Monomeric Clathrin Assembly Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180; D005982:Glutathione Transferase