QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 March 2004
doi: 10.1242/jcs.01062

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Barth, M. Articles by Holstein, S. E. H. Search for Related Content PubMed PubMed Citation Articles by Barth, M. Articles by Holstein, S. E. H. Social Bookmarking                         What's this?

Identification and functional characterization of Arabidopsis AP180, a binding partner of plant C-adaptin

University of Heidelberg, Department of Cell Biology, Heidelberg Institute for Plant Sciences, 69120 Heidelberg, Im Neuenheimer Feld 230, Germany

View larger version (16K): [in a new window]   Fig. 1. Features of Arabidopsis C-adaptin constructs. (A) At-C-Ad compared with its mammalian ortholog. GST-fusion proteins of At-C-Ad comprising either the full ear region (residues 536-1013; GST-At-C-h-ear) or its truncated version (residues 536-759; GST-At-C-h-ear). (B) Three-dimensional structure modelling of the hinge ear region of At-C-Ad was visualized using the Rasmol program, version 2.6. (C) Distribution of the specific binding sites within the two subdomains of mouse and Arabidopsis C-adaptin ear regions. (D) Alignment of the three C-ear binding-sites. Amino acid exchanges are underlined. Note the high degree of identity. (E) Coomassie-stained gels of the purified GST-fusion protein constructs of the At-C-Ad hinge ear region. (1) GST-At-C-h-ear, (2) GST-At-C-h-ear marked by a star and (3) control GST.

View larger version (46K): [in a new window]   Fig. 2. Binding of mammalian network proteins to plant C-adaptin ear region. Pull-down experiments using pig brain cytosol. Incubation with the respective antibodies shows that the proteins bind exclusively to the full-length ear construct but neither to the truncated version nor to the GST control. Note that epsin does not bind. P, pellet; S, supernatant.

View larger version (24K): [in a new window]   Fig. 3. Features of AP180 homologs and At-AP180 constructs. (A) Full-length At-AP180 comprises an N-terminal ENTH-domain and two putative binding sites for the -adaptin ear region (473DPW, 608DPF), as well as one clathrin-binding consensus motif (442DLL). The histidine or GST-fusion protein constructs of At-AP180, lacking the ENTH domain (H6/GST-At-AP180ENTH), were used in pull-down and in clathrin assembly experiments. (B) Commassie-stained gel of the purification of the histidine-tagged recombinant full-length At-AP180 protein (predicted Mr is 72x103; lane 1, marked by a star) and also consecutive stepwise purification of the H6-At-AP180ENTH fusion-protein (predicted Mr is 34.5x103) after incubation with Ni-NTA-beads (2) then boiled (3) and then after gelfiltration on Superose 6/FPLC (4). The purified GST-AP180ENTH fusion-protein has a molecular mass of 55 kDa (5).

View larger version (32K): [in a new window]   Fig. 4. Plant AP180ENTH binding to C-adaptin ear constructs. (A) Pull-down experiments with the full-length ear region or the truncated version of At-C-Ad and GST as a control together with the recombinant H6-AtAP180ENTH construct show that binding requires the ear region. (B) Analysis of the consensus binding motifs of At-AP180 for binding to the C-ear. Binding experiments with alanine substitutions mutans of either the DPF motif (At-AP180ENTH-DPF/AAA) or the DPW-motif (At-AP180ENTH-DPW/AAA) with the full ear construct of At-C-adaptin reveal that the DPF-motif is crucial for binding. GST serves as a control. The antibody against histidine recognizes the AP180-constructs in immunoblots of (A) and (B). P, pellet; S, supernatant.

View larger version (19K): [in a new window]   Fig. 5. Binding of At-C-Ad and At-AP180 measured by plasmon resonance (BIAcore 3000). The full-length ear construct and the truncated version of At-C-Ad were immobilized on CM-5 chips and H6-At-AP180ENTH was passed over the sensor surface for 1 minute (association) followed by a 1 minute wash with running buffer (dissociation). (A) The full ear construct binds H6-At-AP180ENTH with high affinity (kD=35 nM) compared with the truncated version At-C-hear (kD=682 nM). (B) The binding between GST-At-C-h-ear and H6-At-AP180ENTH is concentration dependent as shown by increasing amounts of the At-AP180 construct.

View larger version (12K): [in a new window]   Fig. 6. At-AP180 is a clathrin binding protein. Pull-down assay with clathrin from Arabidopsis cytosol. The clathrin heavy-chain polypeptide (CHC, 180 kDa) is present in the pellet (P) after incubation with GST-At-AP180ENTH. Mutation of the clathrin binding-motif DLL for alanine (DLL/AAA) did not abolish binding; GST served as the control. Immunoblot with the anti-CHC specific antibody.

View larger version (38K): [in a new window]   Fig. 7. At-AP180 functions as a clathrin assembly-protein. (A) Gefiltration (Superose 6/FPLC) of coat proteins removed from pig brain CCV. The respective antibodies were used to show the separation of endogeneous AP180 in fractions 26-30 from clathrin triskelia in fractions 17-25. (B) Reassembly experiments. Successful assembly of clathrin triskelia occurs only in the presence of plant H6-At-AP180ENTH. Assembly is abolished when DLL is exchanged into alanine (GST-At-AP180ENTH-DLL/AAA in a 1:1 stoichiometry). For reassembly, clathrin was incubated either alone or in the presence of half-molar (2:1) and equimolar (1:1) amounts of H6-At-AP180ENTH, and also in its threefold excess (1:3). H6-At-AP180ENTH (DLL) in the same amount as in (1:3) was also incubated alone to show that no unspecific aggregates were present in the pellets (P). Distribution of CHC and the fusion-protein constructs of At-AP180 in (1:1), (1:3), DLL/AAA and DLL are shown on a Coomassie-stained gel and on an immunoblot with the CHC antibody. S, supernatant.

View larger version (104K): [in a new window]   Fig. 8. H6-At-AP180ENTH assembles clathrin triskelia into cages of regular size. Electron micrographs (negative staining) of the reassembly experiment shown in Fig. 7. Assembly activity of clathrin alone (A), clathrin reassembled in a threefold excess of H6-At-AP180ENTH (B), and clathrin reassembled in a threefold excess of GST-At-AP180ENTH-DLL/AAA (C). Close-ups of the cages are inserted. Bar, 250 nm in the overviews and 50 nm in the close-ups. (D) Size distribution of assembled cages from (B) is quantified in bar charts. Note the narrow size distribution.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter