A. Human primary T cells were nucleofected with peGFP-N3 (GFP, panel 2), or with peGFP-N3 containing TRAC-1 (panel 3) or C37,40A-TRAC-1 (panel 4) cDNA to express C-terminally tagged GFP fusion proteins. 16 h after transfection, cells were stimulated with plate-bound anti-CD3/28 for 18 h. Untransfected, unstimulated cells were included as controls (panel 1). Cells were stained with anti-CD69-APC, acquired by FACS and gated on live cells. The percentage of cells in each quadrant is indicated.
B. The percentage of CD69+ cells was calculated separately for GFP-negative (fluorescence intensity 0-10), GFP-low (fluorescence intensity 10-100) and GFP-high (fluorescence intensity >100) populations of the experiment in (A).
C. Six experiments were performed as in A. For each transfection, the percentage of CD69+ cells in the GFP+ population was divided by that in the GFP− population (see materials and methods). The averages of these ratios are represented for each of the transfected constructs.
D. Jurkat T cells were transfected with GFP, TRAC-1/GFP or C37,40A-TRAC-1/GFP expressing constructs. 24 h after transfection, GFP+ cells were sorted by MoFlo® and stimulated with plate-bound anti-CD3/CD28 for 48 h. FACS plots of cells stained with anti-CD69-APC are shown.
E. The culture supernatants of sorted Jurkat cells in (D) were analyzed for the presence of IL-2 by ELISA and represented in a graph (right). A representative of one out of two experiments is shown.
F. Bacterially expressed GST fusion proteins of TRAC-1 and C37,40A-TRAC-1 were captured on glutathione-sepharose beads and incubated for 90 min at 30°C with purified E1, His-tagged E2 UbcH5c, ubiquitin and ATP. After the reaction, beads were analyzed by Western blotting with antibodies against ubiquitin to detect ubiquitin ligase activity (top). The reaction mixture was blotted with anti-GST (middle) and anti-His (bottom) to detect the presence of TRAC-1 proteins and UbcH5c, respectively.
G. Yeast was transformed with GAL4 binding domain (BD) and activation domain (AD) containing vectors, either without insert (-) or with TRAC-1 (wildtype or the C37,40A mutant) or UbcH5a as inserts, as indicated. An interaction between proteins results in β-galactosidase activity and was measured with CPRG as substrate.

A. Jurkat T cells were transfected with the indicated TRAC-1/GFP constructs. The cells were either left untreated or were incubated with the proteasome inhibitor MG-132 (50 μM) for 30, 60 or 120 min. Equivalent amounts of cell lysates were analysed by Western blotting with anti-GFP to detect TRAC-1/GFP proteins, or with anti-c-Cbl.
B. HEK293T cells transfected with TRAC-1/myc cDNA were either left untreated or incubated with MG-132 (50 μM) for two hours. The cell lysates were blotted with anti-myc antibodies.
C. Plasmids for myc-tagged TRAC-1, C37,40A-TRAC-1 or G2A-TRAC-1 were cotransfected with HA-ubiquitin into HEK293T cells. TRAC-1 proteins were immunoprecipitated with anti-myc and blotted with anti-ubiquitin antibodies to detect ubiquitination (top). Lysates were blotted with anti-myc to detect the transfected TRAC-1 proteins (bottom).
D. COS-7 cells were transfected in suspension with myc-tagged TRAC-1 or C37,40A-TRAC-1 and equal cell numbers were divided over five dishes. Cells were incubated the next day with medium alone (0) or with medium containing cycloheximide (100 μg/ml) for the indicated times. Equal amounts of cell lysates were analyzed by Western blotting with anti-myc antibody.
E. Densitometry of the blots shown in D. For each time point, the density of the TRAC-1 band following subtraction of background levels and equalized for a loading control were determined. TRAC-1 levels are expressed as a percentage of levels in untreated cells.

A. HEK293T cells were transfected with plasmids expressing HA-ubiquitin and either myc-tagged wildtype TRAC-1, empty vector (-), TRAC-1ΔC, TRAC-1Δ205 or V217P,S221Q-TRAC-1. TRAC-1 proteins were immunoprecipitated with anti-myc and blotted with anti-HA to detect ubiquitination (top). Ub: ubiquitinated TRAC-1; Ig HC and Ig LC: the heavy chain (HC) and the light chain (LC) of the immunoprecipitating anti-myc antibody, respectively. The same blot was re-probed with anti-myc antibody to detect expression of TRAC-1 proteins (bottom).
B. Comparison of the consensus sequence for the UIM domain [according to [47]] with the TRAC-1 sequence of amino acids 210 - 224. The notation is as follows: e, negatively charged; X, helix favouring; Φ, hydrophobic, z, bulky hydrophobic or polar amino acid.
C. HEK293T cells were transfected with plasmids expressing HA-ubiquitin together with either TRAC-1/myc, TRAC-1/GFP or GFP. Proteins were immunoprecipitated with anti-myc (lane 1) or anti-GFP (lanes 2 and 3) and blotted with anti-HA to detect ubiquitination (top). The lysates were blotted for GFP (bottom).
D. GST fusion proteins were isolated on glutathione-sepharose beads and incubated with lysates from HA-ubiquitin transfected HEK293T cells. Beads were analysed by western blotting with anti-HA antibodies to detect the binding of ubiquitinated proteins to the recombinant proteins (top). TRAC-UIM: amino acids 181 – 232 of TRAC-1 fused to the C-terminus of GST; V/S-UIM: amino acids 181 – 232 with point mutations V217P and S221Q fused to the C-terminus of GST. GST itself was similarly purified and used as a control. To detect levels of expression for TRAC-UIM and V/S-UIM the blot was probed with anti-GST antibodies (middle). This is not shown for GST because of the high expression levels of this protein. The expression of GST, relative to the other constructs, is shown on a Coomassie stained gel (bottom).
E. GST, and GST fusion proteins as in (D) were isolated on glutathione beads and incubated with purified K48-linked poly-ubiquitin_2-7 chains. Beads alone (-) were included as a control for specific binding. Beads were analysed for binding of ubiquitin with anti-ubiquitin antibodies (top). Input K48 poly-ubiquitin_2-7 was analysed on the same polyacrylamide gel but a shorter exposure of the blot is shown. Beads were blotted with anti-GST antibodies to reveal relative levels of the proteins (bottom).
F. HeLa cells were transfected with constructs expressing myc-tagged wildtype TRAC-1, TRAC-1ΔC, TRAC-1Δ205 or V217P,S221Q-TRAC-1. 24 hours after transfection, cells were stained after with anti-myc antibodies and observed by confocal micrsocopy Scale bar: 10 μM.
G. HEK293T cells transfected with TRAC-1Δ205/myc were homogenized in hypotonic buffer and fractionated on discontinuous 70%/65%/10% sucrose gradients as in Fig. 2B. Fractions were collected from the top and aliquots were analyzed by immunoblotting with anti-myc antibodies. Membranes float up to the 65%/10% sucrose interface (fraction 3), whereas soluble proteins remain at the bottom (fractions 10 and 11).

A. HEK293T cells were transiently transfected with TRAC-1/myc constructs and grown in the presence of ³H-myristic acid for 4 hours. TRAC-1/myc proteins were immunoprecipitated with anti-myc antibodies. One half of the sample was analyzed by SDS-PAGE and autoradiography (top), the other half by immunoblotting with anti-myc antibody (bottom).
B. HEK293T cells transfected with TRAC-1/myc constructs were homogenized in hypotonic buffer and fractionated on discontinuous 70%/65%/10% sucrose gradients. Fractions were collected from the top (fraction 1), and aliquots were analyzed by immunoblotting with anti-myc antibodies to detect TRAC-1 proteins, as well as with antibodies against Hsp90, a soluble protein, and Transferrin Receptor (TFR), a transmembrane protein.
C. COS-7 cells transfected with GFP and either TRAC-1/myc or G2A-TRAC-1/myc cDNAs were grown on 13 mm glass coverslips. 24 hours after transfection, cells were permeabilized, fixed, and stained with anti-myc as primary and rhodamine-conjugated goat-anti-mouse as secondary antibody. Confocal images of single channels as well as overlap images are shown. Scale bar: 10 μm. A cytofluorogram (right panels) is represented to show the intensities of red and green fluorophores per pixel. The relative amount of colocalization was determined using cross correlation analysis and the values (with 0 representing no colocalization, and 1 complete colocalization) are shown in the cytofluorgram.
D. COS-7 cells were transfected with TRAC-1/myc and a plasmid for UD-GFP, a myristoylated and palmitoylated protein that contains the unique domain of Lck fused to the N-terminus of GFP. Cells were stained, observed and analyzed as in (C). Scale bar: 10 μm.
E. HeLa cells were transfected with TRAC-1/myc and stained with anti-myc and anti-GM130, a cis-Golgi marker. Scale bar: 10 μm.

A. Amino acid alignments of the human proteins TRAC-1 (Q96EQ8), RNF114 (Q9Y508), RNF138 (Q8WVD3) and RNF166 (Q96A37). Alignments were performed using vector NTI software (Invitrogen). Identical residues are highlighted in black, conserved hydrophobic resides in light grey, other conservative changes in darker grey. Star symbols indicate the conserved Cys and His residues in the various domains. The arrows indicate amino acids in TRAC-1 that have been mutated in this study and the triangles show the positions where truncations have been made.
B. Schematic diagram of the domain composition of TRAC-1 and its family members. Indicated are the myristoylation site (m, present in TRAC-1 only) the RING domain, the newly identified C2HC and C2H2 zinc binding motifs and the UIM domain. The numbers refer to amino acid positions in TRAC-1. The gene composition with the exon boundaries relative to the protein domains is depicted below. RNF138 contains one extra exon compared to the other three proteins.