Sequence analysis of Arabidopsis thaliana E/ANTH-domain-containing proteins: membrane tethers of the clathrin-dependent vesicle budding machinery.

The epsin N-terminal homology (ENTH) domain is a conserved protein module present in cytosolic proteins which are required in clathrin-mediated vesicle budding processes. A highly similar, yet unique module is the AP180 N-terminal homology (ANTH) domain, which is present in a set of proteins that also support clathrin-dependent endocytosis. Both ENTH and ANTH (E/ANTH) domains bind to phospholipids and proteins, in order to support the nucleation of clathrin coats on the plasma membrane or the trans-Golgi-network membrane. Therefore, E/ANTH proteins might be considered as universal tethering components of the clathrin-mediated vesicle budding machinery. Since the E/ANTH protein family appears to be crucial in the first steps of clathrin-coated vesicle budding, we performed data base searches of the Arabidopsis thaliana genome. Sequence analysis revealed three proteins containing the ENTH signature motif and eight proteins containing the ANTH signature motif. Another six proteins were found that do not contain either motif but seem to have the same domain structure and might therefore be seen as VHS-domain-containing plant proteins. Functional analysis of plant E/ANTH proteins are rather scarce, since only one ANTH homolog from A. thaliana, At-AP180, has been characterized so far. At-AP180 displays conserved functions as a clathrin assembly protein and as an alpha-adaptin binding partner, and in addition shows features at the molecular level that seem to be plant-specific.

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.

Arabidopsis mu A-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1.

In receptor-mediated transport pathways in mammalian cells, clathrin-coated vesicle (CCV) mu-adaptins are the main binding partners for the tyrosine sorting/internalization motif (YXXØ). We have analyzed the function of the mu A-adaptin, one of the five mu-adaptins from Arabidopsis thaliana, by pull-down assays and plasmon resonance measurements using its receptor-binding domain (RBD) fused to a histidine tag. We show that this adaptin is able to bind the consensus tyrosine motif YXXØ from the pea vacuolar sorting receptor (VSR)-PS1, as well as from the mammalian trans-Golgi network (TGN)38 protein. Moreover, the tyrosine residue was revealed to be crucial for binding of the complete cytoplasmic tail of VSR-PS1 to the plant mu A-adaptin. The trans-Golgi localization of the mu A-adaptin strongly suggests its involvement in Golgi- to vacuole-trafficking events.

Clathrin and plant endocytosis.

Endocytosis requires the coordinated interaction of a plethora of cytosolic and membrane proteins. In mammalian cells, clathrin plays a crucial role in this process as a scaffolding protein underlying the invaginating plasma membrane and surrounding the primary endocytic vesicle. Despite great similarities at the morphological level, the cargo of endocytic clathrin-coated vesicles in plant cells remains to be elucidated. Thus, the role of endocytosis in the plant cell is difficult to ascertain. This review will present important discoveries on putative endosomal compartments and on the functions of plasma membrane-derived plant clathrin-coated vesicles, but will also emphasize the striking similarities of the clathrin-, network- and vesicle fusion-machineries between plant and animal cells.

Functional evidence for the identification of an Arabidopsis clathrin light chain polypeptide.

Clathrin light chains (CLCs) are regulatory subunits of clathrin triskelia. Based on homology searches in Arabidopsis thaliana data bases we have identified three putative CLC clones, and have focused on the one with the highest homology to mammalian CLC sequences. Analysis of its sequence has revealed coiled-coil structures within a region that corresponds to the clathrin heavy chain-binding site. In addition there is a stretch of acidic amino acids, which is required for the regulatory function of CLC in clathrin assembly. This putative plant CLC ortholog, expressed in bacteria as a glutathione-S-transferase- and myc-tagged fusion protein, was shown to bind to CLC-free recombinantly expressed mammalian clathrin hubs. In contrast, purified native mammalian triskelia with endogeneous CLC did not bind the recombinant putative plant CLC. Based on the conserved sequences between the three Arabidopsis candidates it appears that plants, unlike mammals, may have more than two CLCs.