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

Abstract

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.

FIG. 1

Immunochemical analysis of LRPPRC antigen in interphase cells. COS7 (A–D) and HepG2 (E) cells were cultured, fixed, and incubated with mouse monoclonal antibody Mab4C12 raised against LRPPRC as described in the Materials and Methods. The mouse antibody was detected by Texas Red-X–labeled goat anti-mouse lgG (red), and nuclei were stained with DAPI (blue). Colors from the left and middle panels were merged in the right panel. Pink indicates overlap of the red LRPPRC antibody stain with the blue DAPI DNA stain. Bar upper left, 10 μm. LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex; DAPI, 4,6-diamidino-2-phenylindole; DNA, deoxyribonucleic acid; HepG2, human hepatoma cells.

FIG. 2

Analysis of LRPPRC antigen in mitotic HepG2 cells. The percentage of mitotic HepG2 cells in the cultured population was increased by serum depletion and subsequent addition of 5% serum for 4 h (Materials and Methods). The antigen LRPPRC (red) and DNA (green) were analyzed as described in Fig. 1. Bar upper left, 10 μm. LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex; DNA, deoxyribonucleic acid; HepG2, human hepatoma cells.

FIG. 3

Colocalization of LRPPRC antigen with mitochondria in interphase cells. HepG2 (A) and COS7 (B) cells were labeled for 30 min with 1 μM of MitoTracker® Red CM-H₂XRos (assigned green) before fixation and analysis for LRPPRC with Mab4C12 and FITC-conjugated goat anti-mouse-IgG antibody (assigned red). COS7 cells were treated with 10 μM taxol or nocodazole overnight before the above analysis as indicated. Merged yellow indicates colocalization of LRPPRC antigen and mitochondria. Bar upper left, 10 μm. LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex; HepG2, human hepatoma cells; FITC, fluorescein isothiocyanate.

FIG. 4

Colocalization of recombinant GFP-labeled LRPPRC, CECR2B, UXT, and C19ORF5 with native LRPPRC antigen. COS7 cells were transiently transfected with cDNAs coding for the indicated products labeled with GFP at the N terminus. Cells were stained with Mab4C12 antibody as described in Fig. 1. Product labeled with GFP is indicated in green and Mab4C12-stained antigen in red. Orange and yellow indicate overlap of GFP-labeled product and LRPPRC antigen. Bar upper left, 10 μm. GFP, green fluorescent protein; LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex; cDNA, complementary deoxyribonucleic acid.

FIG. 5

Nuclear condensation of GFP–C19ORF5 and disintegration of DNA. COS7 cells were fixed 72 h after transient transfection of GFP–C19ORF5 and counterstained with DAPI. (A) Binucleate cell with condensed GFP–C19ORF5 in one nuclei. a and b are the same fields viewed at a 20-times different sensitivity setting. (B) Examples of nuclear condensation of GFP–C19ORF5 coincident with disintegration of DNA. The relative intensity of the GFP signal was 1, 2, 10, and 20 in examples I, II, III, and IV, respectively, whereas the DAPI (DNA) signal intensity was uniform. Individual green GFP and blue DAPI fluorescence is indicated as white and merged, respectively. Bar upper left, 10 μm. GFP, green fluorescent protein; DNA, deoxyribonucleic acid; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 6

Association of condensed GFP–CECR2B with condensed DNA. COS7 cells were fixed 72 h after transfection with GFP–CECR2B (green) and stained with DAPI (blue). Four different examples in which condensed DNA appears in distinct foci in different parts of the nucleus are indicated. Cyan in the right panel indicates overlap of the two signals. Bar upper left, 10 μm. GFP, green fluorescent protein; DNA, deoxyribonucleic acid; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 7

Colocalization of GFP-labeled LRPPRC, UXT, and C19ORF5 with the β-tubulin. COS7 cells were transiently transfected with the GFP-labeled product indicated by green and then stained with mouse anti-βtubulin (clone TUB 2.1) monoclonal antibody followed by goat Texas Red–conjugated anti-mouse-IgG antibody (red). Nuclei were stained with DAPI (blue). Colors were merged in the right panel, where yellow indicated overlap of GFP-labeled products with β-tubulin. Cyan indicated overlap of nuclear-associated green GFP with blue DAPI–stained DNA. Bar upper left, 10 μm. GFP, green fluorescent protein; LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 8

Lack of colocalization of GFP-labeled LRPPRC, UXT, and C19ORF5 proteins with the actin cytoskeleton. Cells expressing the same GFP-labeled expression products (green) described in Fig. 7 were permeabilized as described in the Materials and Methods. F-actin was stained with Texas Red-X–tagged phalloidin (red). Nuclear DNA was stained with DAPI (blue). In the merged panel, absence of significant yellow indicates lack of overlap of GFP-products and F-actin. Cyan indicates overlap of GFP and DAPI signals. Image capture was unfiltered to avoid an interfering brown background at 488 nm. This resulted in some artifactual green background in the left panels and consequently some yellow in the merged panel. Bar upper left, 10 μm. GFP, green fluorescent protein; LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex; DNA, deoxyribonucleic acid; DAPI, 4,6-diamidino-2-phenylindole.

FIG. 9

Potential identity, interactions, and function of the LRPPRC complex. Potential interactions in interphase cytosol and nucleus plus mitotic metaphase and anaphase are indicated. Component acronyms are defined in the text, in Table 1, and below. Shaded components indicate a strictly nuclear location in interphase cells. A separated sister chromatid attached to a microtubule (MT) at the kinetochore (K) is depicted in the mitosis schematic. TFIID, subunit of TATA-binding transcription initiation factor; RNAPII, RNA polymerase II; CTD, the C-terminal domain of the large subunit of RNA polymerase I; LRPPRC, leucine-rich pentatricopeptide repeat-motif–containing complex.