Characterization of 2c18 and its interaction with γ1-adaptin. (A) Fixed (left panel) and living (right panel) Vero cells expressing GFP-tagged 2c18 showing the protein localizing to the juxta-nuclear Golgi region, punctate and tubular structures (arrows) and the plasma membrane (arrowheads). Bars represent 10 μm. A time-lapse video showing the dynamics of 2c18-GFP is available as Supplementary data (video 1). (B) Amino-acid sequence of the 2c18 ORF. Residues in gray represent the predicted coiled-coil domain, dotted underlined residues indicate a predicted tyrosine kinase phosphorylation site and solid underlined residues designate the peptide sequence used to generate the anti-2c18 antibodies. Bold residues show potential GAE-binding motifs. (C) Protein extracts prepared from HeLa cells were immunoprecipitated with anti-2c18 antibodies followed by Western blotting for different components of various adaptor complexes as indicated. Only co-immunoprecipitation of γ1-adaptin by 2c18 was detectable, and to a higher degree from 2c18-GFP-transfected cells. No co-immunoprecipitation of α-adaptin, δ-adaptin, GGA1 or GGA3 was detected under these conditions. Lane 1 contains 20 μg of total HeLa extract from nontransfected cells. (D) Various purified GST fusion proteins were incubated with immobilized His-tagged 2c18 and subsequently separated by centrifugation into pellet (P) and soluble (S) fractions to determine binding to 2c18. Following SDS–PAGE and Western blotting, the samples were probed with anti-GST antibodies. Only the γ1-adaptin and GGA1 ‘ear' domains interact with recombinant 2c18. (E) 2c18 peptides containing putative GAE-binding motifs, FGLL (aa 5–18), FENL (aa 35–55), YSGF (aa 270–287), and their counterparts containing alanine substitutions were incubated with GST-γ1-adaptin and GST-GGA1 ‘ears'. All the wild-type peptides bound strongly to the γ1-adaptin ‘ear', but less so to the GGA1 ‘ear'. None of the peptides containing the alanine substitutions bound to either of the GST-tagged proteins. (F) GST-γ1-adaptin ‘ear' was bound to immobilized 2c18 and then competitively eluted with either purified GST-EpsinR or GST alone. The remaining 2c18-bound γ1-adaptin ‘ear' was determined by SDS–PAGE and Western blotting. EpsinR protein was able to competitively remove the γ1-adaptin ‘ear' from 2c18.

2c18 expression levels influence γ1-adaptin membrane association. (A) HeLa cells were immunostained for endogenous 2c18 and γ1-adaptin. Colocalizing structures are marked by arrows. (B) Membrane (mb) and cytosol (cyt) fractions were prepared from HeLa cell extracts and then subjected to SDS–PAGE followed by Western blotting for γ1-adaptin. (C) HeLa cells were treated with siRNA oligonucleotides against 2c18 and then fixed and double stained for 2c18 and γ1-adaptin. Cells showing a decreased 2c18 staining are marked with asterisks. Arrowheads point to γ1-adaptin's juxta-nuclear localization. (D) Vero cells were transfected with a plasmid encoding 2c18-GFP for 18 h and then fixed and stained for γ1-adaptin. Overexpression of 2c18-GFP causes a more compact staining of γ1-adaptin, which now almost totally overlaps with the 2c18-GFP (arrows). Transfected cells are denoted with asterisks. Bars represent 10 μm.

Analysis of 2c18 variants. (A) Protein extracts prepared from HeLa cells, transfected with either 2c18-CFP, 2c18ΔBM-CFP, 2c18 splice-CFP or CFP alone, were immunoprecipitated with anti-GFP/CFP antibodies and blotted for γ1-adaptin. The construct 2c18ΔBM is comprised of full-length 2c18 with the three putative GAE-binding motifs mutated as described in Figure 1. (B) Membrane (mb) and cytosol (cyt) fractions were prepared from HeLa cell extracts expressing various 2c18 constructs as described and then subjected to SDS–PAGE followed by Western blotting for γ1-adaptin. (C) Vero cells expressing 2c18 splice-CFP were fixed and immunostained for γ1-adaptin (upper panel) or treated with 5 μg/ml BFA for 2 min prior to immunostaining (lower panel). Bar represents 10 μm. (D) Purified GST fusion proteins were incubated with immobilized His-tagged 2c18 splice variant or 2c18 coiled-coil domain (aa 78–138) and subsequently separated by centrifugation into pellet (P) and soluble (S) fractions to determine binding to the 2c18 variants. Following SDS–PAGE and Western blotting, the samples were probed with anti-GST antibodies.

Retention of the MPR at the TGN in 2c18-overexpressing cells. (A–C) HeLa cells were transfected with 2c18-CFP, treated with nocodazole (20 μg/ml) for 2 h and then fixed and immunostained for MPR (red) and either GM130 (A) as a cis Golgi marker, p230 (B) as a trans Golgi marker or EEA1 (C) as an early endosome marker (all green). The insets show an enlarged area of the transfected cells in each case. Arrows mark colocalizing structures and arrowheads those that do not colocalize. Transfected cells are denoted with asterisks. Bars represent 10 μm.

Ultrastructural localization of 2c18. (A, C, D) Double labeling of HeLa SA:48 cells for the TGN marker sialyltransferase (12 nm gold, arrowheads) and endogenous 2c18 (15 nm gold, arrows). 2c18 localizes in close proximity to the Golgi complex (G) and the TGN (TGN), in addition to endocytic structures (E). No labeling is detectable on mitochondria (M). (B) The 5 nm gold-BSA (arrowheads) was internalized for 10 min and chased for 20 min to label late endosomes. 2c18 (10 nm gold, arrows) is present on the inner membranes of this multivesicular structure. (E) 2c18 (10 nm gold, arrows) is also seen to localize to the plasma membrane (PM). (F, G) Double labeling of rat pancreatic tissue for γ1-adaptin (10 nm gold, arrowheads) and endogenous 2c18 (15 nm gold, arrows). Both markers are seen to colocalize on TGN buds and vesicular profiles. ‘SG' denotes secretory granules. Bars represent 200 nm.

2c18 overexpression stabilizes AP-1 on membranes in the presence of BFA. Vero cells expressing 2c18-GFP were treated with 5 μg/ml BFA for increasing lengths of time as indicated and then fixed and immunostained for γ1-adaptin and other coat complex components. (A) AP-1 but not COPI retains its membrane localization in cells overexpressing 2c18-GFP after 2 min of BFA treatment. (B) After 30 min BFA treatment, AP-1 is still present on membranes of 2c18-GFP cells. (C) AP-3 and (D) GGA3 are not stabilized on membranes by 2c18-GFP in the presence of BFA. Transfected cells are denoted with asterisks. Bars represent 10 μm.

MPR trafficking in 2c18-knockdown or 2c18-overexpressing cells. (A) Vero cells expressing 2c18-GFP were fixed and stained for MPRs and γ1-adaptin. Arrows indicate colocalizing structures and arrowheads show juxta-nuclear staining of the MPRs. (B) Enlarged view of the juxta-nuclear region of the transfected cell, showing all three markers colocalizing to the same structures (arrows). Bars represent 10 μm. (C) HeLa cells were transfected with a plasmid encoding 2c18-GFP. After 24 h, the maturation and secretion of cathepsin D was determined as described in Materials and methods. ‘C' indicates the cathepsin D recovered from total cell extracts and ‘M' that recovered from the cell culture medium. Arrowheads mark mature cathepsin D and asterisks precursor cathepsin D. Numbers at the bottom of (C, D) indicate the amount of cathepsin D from the ‘M lane' (secreted cathepsin D) divided by the sum of cathepsin D secreted and processed inside cells to the mature form (indicated by an arrowhead in C lane). (D) HeLa cells were transfected with siRNAs as described in Materials and methods. The maturation and secretion of cathepsin D was analyzed as described above.