The tripartite motif family identifies cell compartments

Abstract

A functional genomic approach, based on systematic data gathering, was used to characterize a family of proteins containing a tripartite motif (TRIM). A total of 37 TRIM genes/proteins were studied, 21 of which were novel. The results demonstrate that TRIM proteins share a common function: by means of homo-multimerization they identify specific cell compartments.

Fig. 1. Schematic outline of the strategy used in the first phase to identify and clone full-length TRIM cDNAs and in the second phase to find common functional features through systematic data gathering. Hs, Homo sapiens; Mm, Mus musculus.

Fig. 2. The TRIM protein family. (A) Schematic representation of the TRIM proteins identified in the screen described in Figure 1. Color coding is as follows: red squares, RING domain; light blue squares, B-box type 1 domain; dark blue squares, B-box type 2 domain; marine blue squares, B-box of TRIM alternatively spliced isoforms not following type 1 or 2 criteria; green rectangles, CC region; orange rectangles, NHL repeats domain; yellow rectangles, RFP-like domain regions; brown squares, TSS domain; purple squares, PHD domain; magenta rectangles, bromodomain; black squares, ARF domain. (B) Alignments of the RING, the B-box type 1 and the B-box type 2 domains. The consensus sequences are indicated above. Conserved and similar amino acids are boxed. TRIM9, 24 and 36 double backslashes indicate a discontinuity in the sequence. Abbreviations are as in Figure 1. R, B1 and B2 consensi were built with 33, 14 and 35 mammalian sequences, respectively. Orthologous sequences were omitted for obvious reasons. Please note that TRIM37 in the R domain and TRIM19 and 23 in the B2 domain do not respect the spacing between conserved residues.

Fig. 2. The TRIM protein family. (A) Schematic representation of the TRIM proteins identified in the screen described in Figure 1. Color coding is as follows: red squares, RING domain; light blue squares, B-box type 1 domain; dark blue squares, B-box type 2 domain; marine blue squares, B-box of TRIM alternatively spliced isoforms not following type 1 or 2 criteria; green rectangles, CC region; orange rectangles, NHL repeats domain; yellow rectangles, RFP-like domain regions; brown squares, TSS domain; purple squares, PHD domain; magenta rectangles, bromodomain; black squares, ARF domain. (B) Alignments of the RING, the B-box type 1 and the B-box type 2 domains. The consensus sequences are indicated above. Conserved and similar amino acids are boxed. TRIM9, 24 and 36 double backslashes indicate a discontinuity in the sequence. Abbreviations are as in Figure 1. R, B1 and B2 consensi were built with 33, 14 and 35 mammalian sequences, respectively. Orthologous sequences were omitted for obvious reasons. Please note that TRIM37 in the R domain and TRIM19 and 23 in the B2 domain do not respect the spacing between conserved residues.

Fig. 3. TRIM expression pattern during mouse embryonic development. TRIM2 is preferentially expressed in the central nervous system and gut. (A and B) Sagittal sections of E12.5 and E14.5 embryos, respectively. (C) A coronal section of an E12.5 embryo. All three panels reveal high expression of TRIM2 in the nervous system (in particular in the telencephalon, cranial and dorsal root ganglia, and eye) and in the gut (midgut and duodenum). Higher magnification panels highlight expression of TRIM2 in the neural tube and in the dorsal root ganglia (D) and in the inner neural layer of the retina (E). (F and G) Expression in the adult brain, particularly high in the hippocampus. TRIM8 is mainly expressed in the kidney, gut and central nervous system. Coronal and sagittal sections of E10.5 and E12.5 embryos (H and I) show expression in the central nervous system. The higher magnification panels show a high level of expression at the E14.5 stage in: the eye (lens and inner neural layer of the retina) (J); the primitive glomeruli of the developing kidney (K); the villi of the gut (L); and the dorsal root ganglia (M).

Fig. 4. TRIM proteins homomultimerize and associate with specific subcellular structures. Subcellular localization of TRIM29 (A), TRIM4 (B), TRIM2 (C), TRIM5 (D), TRIM8 (E), TRIM13 (F), TRIM28 (G) and TRIM9 (H). U2OS and HeLa cells were transfected with plasmids encoding EGFP–TRIM fusions. All TRIM proteins localized to particular subcellular compartments: TRIM29, to cytoplasmic ribbon-like structures; TRIM2, to cytoplasmic filaments; TRIM4, 5 and 9, to ‘cytoplasmic bodies’; TRIM8, to specific nuclear bodies; TRIM13, around the nucleus; and TRIM28, to specific chromatin regions. (I) Interaction-mating matrix of 32 TRIM ORFs fused to the LexA binding domain (bait) or to the B42-SV40 NLS-HA-tag domains (preys). Color coding is as follows: red squares, homodimerization; dark green squares, homodimerization previously reported and detected in the present assay; dark blue squares, homodimerization previously reported and not detected in the present assay; yellow squares, heterodimerization detected in either bait-X/prey-Y or bait-Y/prey-X orientation; light green squares, heterodimerization detected in both bait-X/prey-Y and bait-Y/prey-X orientations; orange squares, heterodimerization previously reported and not detected in either bait-X/prey-Y or bait-Y/prey-X orientation; light blue squares, co-presence without direct interaction in a protein complex reported (Grignani et al., 1996; Cao et al., 1997, 1998; Cainarca et al., 1999; Zhong et al., 1999a). (J) Superose 6 gel filtration of in vitro translated TRIM6, 23, 28 and 30 proteins. Their elution peaks correspond to formation of homomultimeric complexes. Elution of proteins of known molecular weight (kDa) is indicated above.

Fig. 5. Characterization of TRIM deletion mutants. (A) Schematic representation of the deletion mutants and the TRIM18 domains. The color coding is as defined in Figure 2A. (B–F) Interaction-mating assays between strains carrying TRIM8 (B), TRIM11 (C) and TRIM29 (D) deletion mutants, TRIM18 domains (F) and the naturally occurring TRIM21α and β isoforms (E). EGY42 bait strains containing plasmids that expressed LexA fusions were mated to EGY48 derivatives that contained B42 fusions. Numbers (1–3) indicate the degree of β-galactosidase activity on GAL plates. No activity was detected for the interaction represented by open squares. Please note that some residual binding is still apparent in the combinations TRIM8ΔCC/TRIM8WT and TRIMΔCC/TRIM8ΔB1. (G and H) Gel filtration of in vitro translated TRIM8 (G) and TRIM11 (H) deletion mutant proteins. The monomeric sizes are as follows. TRIM8: WT, 61 kDa; ΔR, 53 kDa; ΔB, 56 kDa; ΔCC, 50 kDa; and TRIM11: WT, 51 kDa; ΔR, 43 kDa; ΔB, 48 kDa; ΔCC, 40 kDa; ΔRFP, 35 kDa. (I) Co-immunoprecipitation experiments of HA-tagged TRIM18 mutants and Myc-tagged TRIM18 expressed in Cos7 cells.

Fig. 6. Subcellular localization of TRIM6, 8 and 29 deletion mutants.