BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein, TRIM5delta.
We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.
Xu
Lixin
L
Department of Biochemistry and Molecular Biology, F. Edward Hèbert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
Yang
Lihong
L
Moitra
Prasun K
PK
Hashimoto
Keiko
K
Rallabhandi
Prasad
P
Kaul
Sunil
S
Meroni
Germana
G
Jensen
Jane P
JP
Weissman
Allan M
AM
D'Arpa
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D000090103:Antiviral Restriction Factors; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000070584:Tripartite Motif Proteins; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D000090103:Antiviral Restriction Factors; C429757:BTBD1 protein, human; C477345:BTBD2 protein, human; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin; C477346:TRIM5 protein, human; D044767:Ubiquitin-Protein Ligases