Novel complex integrating mitochondria and the microtubular cytoskeleton with chromosome remodeling and tumor suppressor RASSF1 deduced by in silico homology analysis, interaction cloning in yeast, and colocalization in cultured cells.
Availability of the complete sequence of the human genome and sequence homology analysis has accelerated new protein discovery and clues to protein function. Protein-protein interaction cloning suggests multisubunit complexes and pathways. Here, we combine these molecular approaches with cultured cell colocalization analysis to suggest a novel complex and a pathway that integrate the mitochondrial location and the microtubular cytoskeleton with chromosome remodeling, apoptosis, and tumor suppression based on a novel leucine-rich pentatricopeptide repeat-motif-containing protein (LRPPRC) that copurified with the fibroblast growth factor receptor complex. One round of interaction cloning and sequence homology analysis defined a primary LRPPRC complex with novel subunits cat eye syndrome chromosome region candidate 2 (CECR2), ubiquitously expressed transcript (UXT), and chromosome 19 open reading frames 5 (C19ORF5) but still of unknown function. Immuno, deoxyribonucleic acid (DNA), and green fluorescent protein (GFP) tag colocalization analyses revealed that LRPPRC appears in both cytosol and nuclei of cultured cells, colocalizes with mitochondria and beta-tubulin rather than with alpha-actin in the cytosol of interphase cells, and exhibits phase-dependent organization around separating chromosomes in mitotic cells. GFP-tagged CECR2B was strictly nuclear and colocalized with condensed DNA in apoptotic cells. GFP-tagged UXT and GFP-tagged C19ORF5 appeared in both cytosol and nuclei and colocalized with LRPPRC and beta-tubulin. Cells exhibiting nuclear C19ORF5 were apoptotic. Screening for interactive substrates with the primary LRPPRC substrates in the human liver complementary DNA library revealed that CECR2B interacted with chromatin-associated TFIID-associated protein TAFII30 and ribonucleic acid splicing factor SRP40, UXT bridged to CBP/p300-binding factor CITED2 and kinetochore-associated factor BUB3, and C19ORF5 complexed with mitochondria-associated NADH dehydrogenase I and cytochrome c oxidase I. C19ORF5 also interacted with RASSF1, providing a bridge to apoptosis and tumor suppression.
Liu
Leyuan
L
Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Boulevard, Houston, Texas 77030, USA.
Amy
Vo
V
Liu
Guoqin
G
McKeehan
Wallace L
WL
D006528:Carcinoma, Hepatocellular; D018929:Cell Culture Techniques / Q000379:methods; D002874:Chromosome Mapping; D002888:Chromosomes, Human, Pair 19; D003599:Cytoskeleton / Q000648:ultrastructure*; D017930:Genes, Reporter; D015894:Genome, Human; D049452:Green Fluorescent Proteins; D006801:Humans; D008113:Liver Neoplasms; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000648:ultrastructure*; D008928:Mitochondria / Q000648:ultrastructure*; D009842:Oligopeptides / Q000494:pharmacology*; D016366:Open Reading Frames; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D009842:Oligopeptides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins