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The role of auxilin in the uncoating of clathrin-coated vesicles was analyzed by utilizing glutathione-S-transferase (GST)-auxilin fragments. The cofactor activity was detected in a recombinant auxilin fragment with J domains located in segments 813-910 and clathrin-binding domains located in segment 547-814. However, the ability of the recombinant auxilin to assists hsc 70 in the dissociation of clathrin baskets was eliminated when its J domain was truncated at residue 814.

A study has been conducted to examine the role of protein coat auxilin in uncoating clathrin-coated vesicles that transport selected membrane proteins from the clel surface to the endosomal system. The results showed that auxilin binds with high affinity to clathrin lattices to catalyze protein folding and protein transport across the cell membrane. Auxilin has also been shown to catalyze the selective disruption of protein-protein interactions.

Plant orthologs of mammalian-trafficking proteins involved in endocytotic cycling are discussed. The use of styryl dye FM4-64 to define the course of endocytotic uptake and the fungal toxin brefeldin A to dissect the internationalization pathways are particularly emphasized.

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 αadaptin 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 α-adaptin orthologs from Arabidopsis thaliana: AtAP180 and At-αC-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-αC-Ad and the C-terminal region of At-AP180 were used as fusion proteins in pulldown experiments and plasmon-resonance measurements. At-αC-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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Secretion, endocytosis and transport to the lytic compartment are fundamental, highly coordinated features of the eukaryotic cell. These intracellular transport processes are facilitated by vesicles, many of which are small (100 nm or less in diameter) and ‘coated’ on their cytoplasmic surface. Research into the structure of the coat proteins and how they interact with the components of the vesicle membrane to ensure the selective packaging of the cargo molecules and their correct targeting, has been quite extensive in mammalian and yeast cell biology. By contrast, our knowledge of the corresponding types of transport vesicles in plant cells is limited. Nevertheless, the available data indicate that a considerable homology between plant and non-plant coat polypeptides exists, and it is also suggestive of a certain similarity in the mechanisms underlying targeting in all eukaryotes. In this article we shall concentrate on three major types of transport vesicles: clathrin-coated vesicles, COP-coated vesicles, and ‘dense’ vesicles, the latter of which are responsible for the transport of vacuolar storage proteins in maturing legume cotyledons. For each we will summarize the current literature on animal and yeast cells, and then present the relevant data derived from work on plant cells. In addition, we briefly review the evidence in support of the ‘SNARE’ hypothesis, which explains how vesicles find and fuse with their target membrane. [ABSTRACT FROM AUTHOR]

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.