GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2006) 11, 1295-1304. doi:10.1111/j.1365-2443.2006.01017.x
© 2006 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirano, Y.
Right arrow Articles by Takeyasu, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirano, Y.
Right arrow Articles by Takeyasu, K.

Human small G proteins, ObgH1, and ObgH2, participate in the maintenance of mitochondria and nucleolar architectures

Yasuhiro Hirano*, Ryosuke L. Ohniwa, Chieko Wada, Shige H. Yoshimura and Kunio Takeyasu

Graduate School of Biostudies, Kyoto University, Kitashirakawa-ohiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The Obg subfamily protein is one of the P-loop small G proteins and is highly conserved in many organisms from bacteria to human. Two obg genes, obgH1 and obgH2, exist in the human genome. Both ObgH1 and ObgH2 showed similar GTPase activities (0.014 ± 0.005 and 0.010 ± 0.002/min for ObgH1 and ObgH2, respectively) to those of the bacterial Obg proteins and complemented the Obg function in Escherichia coli ribosome maturation, suggesting that the functions of Obg proteins are well conserved through evolution. Immunofluorescence microscopy of HeLa cells revealed that ObgH1 localizes in mitochondria, and ObgH2 in the dense fibrillar compartment region of the nucleolus. Knock-down of ObgH1 by RNAi induced mitochondria elongation, whereas knock-down of ObgH2 resulted in the disorganization of the nucleolar architecture. In conclusion, the two human Obg proteins have similar enzymatic activities that can complement bacterial Obg function, but show different cellular function(s) with different intracellular localizations.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The small G proteins act as molecular switches and play crucial roles in the regulation of fundamental cellular processes, such as protein synthesis, nuclear transport, membrane trafficking, and signal transduction (Bourne et al. 1991). Obg subfamily proteins are such G-proteins and belong to an Obg family that is categorized into Obg-HflX-like super-family within the TRAFAC class in the P-loop GTPase super-class (Leipe et al. 2002).

Database search has demonstrated that Obg subfamily proteins exist in bacteria and eukaryotes, but not in archea (Leipe et al. 2002). In many bacteria, the dysfunction of Obg protein affects cell growth (Morimoto et al. 2002), morphologic differentiation (Okamoto & Ochi 1998), DNA replication (Slominska et al. 2002), and chromosome partitioning (Kobayashi et al. 2001), possibly via a control of ribosome biogenesis (Lin et al. 1999; Scott & Haldenwang 1999; Scott et al. 2000; Lin et al. 2001; Tan et al. 2002; Datta et al. 2004; Lin et al. 2004; Wout et al. 2004; Zhang & Haldenwang 2004; Sato et al. 2005). In Bacillus subtilis, the Obg protein binds to the large ribosomal subunit (50S) protein, L13, and is required for the stress-dependent activation of transcription factor {sigma}B (Scott & Haldenwang 1999; Scott et al. 2000). Similar to the B. subtilis Obg protein, Caulobacter crescentus, Escherichia coli and Vibrio harveyi Obg proteins are also cofractionated with the 50S ribosomal subunit (Datta et al. 2004; Sato et al. 2005; Sikora et al. 2006). In the case of C. crescentus and E. coli, the GTPase activity of Obg protein is required for maintaining the intracellular level of 50S ribosomal subunit (Lin et al. 2001) and Obg-binding to the ribosome (Sato et al. 2005), respectively.

On the other hand, the function of eukaryotic Obg proteins has been studied only in Saccharomyces cerevisiae. Datta et al. have demonstrated that Mtg2p, which is only one S. cerevisiae Obg homolog, carries a putative mitochondria-localization signal at its amino terminus and localizes in the mitochondrial inner membrane. In mitochondria, Mtg2p is cofractionated with mitochondrial large ribosomal subunit (Datta et al. 2005).

Exploring the genomic databases revealed that animals and plants have multiple Obg genes (Fig. 1A). In this report, we investigated the enzymatic function and intracellular localization of the human Obg proteins, named ObgH1 and ObgH2. Amino acid sequence analyses revealed that ObgH1 carries a putative mitochondria-localization signal, but ObgH2 does not, suggesting that ObgH1 and ObgH2 have different cellular functions with different intracellular localizations.


Figure 1
View larger version (42K):
[in this window]
[in a new window]
  		Figure 1  Two Obg subfamily proteins are present in H. sapiens. (A) The number of Obg subfamily genes in a genome is summarized in the left panel. A phylogenetic tree of the Obg subfamily proteins are drawn in the right panel. Bacterial Obg and eukaryotic Obg proteins are depicted in green and red, respectively. The Obg proteins that are aligned in panel B were indicated in blue. (B) Amino acid sequence comparison of Obg proteins. The amino acid sequences of ObgH1, ObgH2, E. coli Obg, and B. subtilis Obg are aligned using CLUSTAL X. The Obg-fold (blue box) and Obg-specific domains (G1–G5, red box) are indicated.

 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Characteristics of primary sequences

A comparison of the amino acid sequences revealed that there are 28% identity and 41% similarity between ObgH1 and ObgH2 (Fig. 1B). Highly conserved sequences clusters in the glycine-rich region (termed Obg-fold; Fig. 1B, blue box) and in the GTPase domain (G1–G5; Fig. 1B, red box) both also exist in other Obg subfamily proteins (Fig. 1B). The prominent difference between ObgH1 and ObgH2 can be seen in the N-terminal regions; only ObgH1 possesses a long N-terminus, which is rich in leucine and lysine residues (19 of 71 amino acids). We named this region as ObgH1 SR (specific region). This region was found to contain a putative mitochondria-localization signal (8Ser-14Val, Fig. 1B, asterisk) by iPSORT prediction (http://psort.nibb.ac.jp).

Enzymatic activity of ObgH1 and ObgH2

Glutathione-S-transferase (GST)-fused full-length ObgH1 and ObgH2 were expressed in E. coli, purified, and subjected to GTPase assays. The kcat values of ObgH1 and ObgH2 were measured to be 0.014 ± 0.005/min (n = 4) and 0.010 ± 0.002/min (n = 15), respectively (Table 1), which are comparable to those of other Obg proteins in bacteria (0.006 in B. subrilis, 0.017 in E. coli, and 0.03 in C. crescentus) (Welsh et al. 1994; Lin et al. 1999; Buglino et al. 2002; Wout et al. 2004).


View this table:
[in this window]
[in a new window]
  		Table 1  GTPase activity of ObgH proteins
 
We then performed complementation assay. We have previously established obgE-deleted E. coli strain (MG1655{Delta}obgE::kan [ori-pSC101 cat repts PBADobgE+]) (Kobayashi et al. 2001; Sato et al. 2005) in which ObgE is supplied from a plasmid harboring ObgE upon arabinose induction, and the plasmid is lost under the nonpermissive temperature. The plasmid that contains the cDNA fragment for ObgH1 or ObgH2 under the control of lac promoter (pTrc99A amp PtrcobgH1 or obgH2) was introduced into this ObgE-deleted E. coli, and incubated under various conditions as described in Experimental procedures. Note that (i) at the nonpermissive temperature, all the cells (even containing ObgH1 and ObgH2) should die in the presence of chloramphenicol as a result of a loss of the ObgE-plasmid, and (ii) if ObgH1 or ObgH2 can complement ObgE function, the cells containing ObgH1 or ObgH2 can survive even after a loss of the ObgE-plasmid at the nonpermissive temperature in the absence of arabinose. At 37 °C in the presence of arabinose, E. coli cells containing the plasmid harboring ObgH1 could not lose the plasmid-harboring ObgE completely, then, the cells could survive in the spot with the highest number of the cells. As can be seen in Fig. 2, the cells harboring ObgH1 or ObgH2 could survive without arabinose at both 30 and 37 °C, although their growth was slower than the cells expressing ObgE.


Figure 2
View larger version (44K):
[in this window]
[in a new window]
  		Figure 2  Both ObgH1 and ObgH2 complement E. coli Obg function(s). (A) The pTrc 99 A plasmids harboring ObgH1 and ObgH2 (PtrcobgH1 or obgH2) were introduced into ObgE-deleted E. coli cells (MG1655{Delta}ObgE::kan [ori-pSC101 cat repts PBADobgE+]). The transformant cells (3 x 103, 3 x 102, 3 x 101 cells for ObgH1 and 9 x 102, 9 x 101, 9 cells for ObgH2, respectively) were spotted on a LB agar plate and incubated at 30 or 37 °C for 3 days under various conditions indicated. These cells were also incubated on a LB plate containing chroramphenicol as a negative control, and did not grow at both 30 and 37 °C (data not shown). (B) The summary of the results in (A).

 
Intracellular localization of ObgH1 and ObgH2 in HeLa cells

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that HeLa cells are expressing both ObgH1 and ObgH2 (Fig. 3A). Immunostaining of HeLa cells with ObgH2-specific antibody showed that ObgH2 localizes in the nucleolus (Fig. 3B). Co-staining with various nucleolar marker proteins 2–30C antigen for the fibrillar center (FC) region (see Experimental procedures for details), fibrillarin for the dense fibrillar component (DFC) region and B23 for the granular component (GC) region revealed that ObgH2 exists in the DFC region (Fig. 3B). ObgH2 dynamically changed its localization throughout the cell cycle. At the onset of mitosis, ObgH2 moved to chromosome surface, and remained until anaphase (Fig. 3C). Finally, ObgH2 gradually re-assembled into the nucleoli at late anaphase to telophase. Co-immunostaining demonstrated that ObgH2 co-localized with Ki-67 antigen throughout mitosis (Fig. 3D).


Figure 3
View larger version (45K):
[in this window]
[in a new window]
  		Figure 3  Intracellar localization of ObgH2 in HeLa cells. (A) RT-PCR detection of ObgH1- and ObgH2-mRNAs. Total RNA was isolated from HeLa cells and RT-PCR was performed using specific primer sets for ObgH1 and ObgH2. (B, C) Intracellular localization of ObgH2. (B) HeLa cells in the interphase were double-stained with antibodies for ObgH2 (green) and nucleolar marker proteins (2–30C, fibrillarin or B23; red). An enlarged image of nucleolus is shown as inset. Bar: 10 µm. (C) Mitotic cells were stained with antibody for ObgH2 (green) and DAPI (blue) and observed with confocal microscopy (left panels) and conventional fluorescence microscopy (right panels). (D) HeLa cells in the mitotic phase were costained with antibodies for ObgH2 (green) and Ki-67 antigen (red). Bar: 10 µm.

 
In contrast, when tagged with V5 or GFP at its C-terminus (ObgH1-V5 and ObgH1-GFP) and expressed in HeLa cells, ObgH1 localized in the mitochondria (Fig. 4A). Biochemical assay using isolated mitochondria from the ObgH1-V5-expressing HeLa cells supported this result (Fig. 4B). To investigate the mechanism of mitochondrial localization of ObgH1, the full-length ObgH1 (corresponding to 1–406 amino acids) fused with GFP at its N-terminus (GFP-ObgH1), ObgH1 SR (corresponding to 1–71 amino acids) fused with GFP at its C-terminus (ObgH1 SR-GFP) and the SR-deleted ObgH1 (72–406 amino acids) fused with GFP at its C-terminus (ObgH1 NC-GFP) were expressed in HeLa cells. As seen in Fig. 4C, ObgH1 SR-GFP localized in the mitochondria as well as ObgH1-GFP, whereas GFP-ObgH1 and ObgH1 NC-GFP did not, demonstrating that ObgH1 SR functions as a mitochondria-localization signal in vivo. Interestingly, a deletion of the putative mitochondria-localization signal ({Delta}8–14) did not affect the mitochondrial-localization of ObgH1 (Fig. 4D). This result suggests that this sequence alone is insufficient as the functional mitochondria-localization signal in vivo. Another deletion mutant, {Delta}1–14, still localized to the mitochondria, but further deletions, {Delta}1–30 and {Delta}1–50, could not, suggesting that a critical mitochondria-localization signal exists in the region between 14th and 30th amino acids.


Figure 4
View larger version (37K):
[in this window]
[in a new window]
  		Figure 4  Intracellar localization of ObgH1 in HeLa cells. (A, B) ObgH1 localizes in the mitochondria. (A) Immunofluorescence microscopy of HeLa cells expressing GFP-tagged ObgH1 or V5-tagged ObgH1. The cells expressing V5-tagged ObgH1 was immunostained with anti-V5 antibody. Mitochondria were visualized with Mitotracker Red CMXRos (red). Bar: 10 µm. (B) Mitochondria were isolated from HeLa cells expressing V5-tagged ObgH1 and subjected to immunoblot analysis using anti-V5 antibody. The purity of isolated mitochondria was analyzed using anti-cytochrome c antibody and anti-GAPDH antibodies. M: mitochondria fraction, C: cytosol fraction. (C) The ObgH1 SR contains a mitochondria-localization signal. The HeLa cells expressing ObgH1 SR-GFP, GFP-ObgH1, or ObgH1 NC-GFP were either directly observed by fluorescence microscopy or observed after staining with Mitotracker Red CMXRos (red). The schematic diagrams of the constructs are summarized in the left panel. Bars: 10 µm. (D) ObgH1 SR-deletion mutants ({Delta}8–14, {Delta}1–14, {Delta}1–30 and {Delta}1–50) fused with GFP were expressed in HeLa cells and directly observed by confocal microscopy. The schematic diagrams of the constructs are summarized in the left panel. Bars: 10 µm.

 
Knock-down of ObgH1 and ObgH2 by RNAi

The specific siRNAs against ObgH1 or ObgH2 were introduced into HeLa cells as described in Experimental procedures. The siRNA against ObgH2 repressed the expression of endogenous ObgH2 around 50% (Fig. 5A). Microscopic observations of the ObgH2-knocked-down cells revealed that the shape of the nucleolus was deformed. The FC marker protein showed several strong dots in or out of the nucleolus (Fig. 5B, arrow) and GC region accumulated at the periphery of the nucleolus (Fig. 5B, double arrowhead). On the other hand, the DFC region was almost unaffected, although some of the fibrillarin signal dispersed throughout the nucleolus and formed a big ring-like structure (Fig. 5B, arrowhead).


Figure 5
View larger version (30K):
[in this window]
[in a new window]
  		Figure 5  Knock-down of ObgH2 induced the breakdown of nucleolar structures. HeLa cells were transfected with siRNA for ObgH2. (A) The total cell lysates from the transfected and control cells were prepared and analyzed by Western blotting using anti-ObgH2 and anti-ß-actin antibodies. (B) The transfected cells were immunostained with antibodies for ObgH2 (green), and nucleolar proteins (2-30C antigen, fibrillarin and B23; red). Enlarged images of nucleolus are shown as insets. Arrows, arrowheads and double arrowheads indicate the localization of the nucleolar proteins. Bar: 10 µm.

 
To verify whether ObgH1-specific siRNA can repress the expression of ObgH1, we introduced this siRNA into HeLa cells and estimated mRNA level of ObgH1 by RT-PCR (Fig. 6A). The amount of ObgH1 mRNA was mostly decreased in the siRNA-treated cells. To examine the knock-down efficiency at the protein level, we introduced ObgH1-specific siRNA into ObgH1-V5 expressing HeLa cells and performed a Western blot analysis using V5-antibody. As seen in Fig. 6B, this ObgH1-specific siRNA could mostly repress the expression of ObgH1-V5 at the protein level. Therefore, we concluded that this siRNA is also able to knock-down endogenous ObgH1 in HeLa cells. Microscopic observation revealed that the ObgH1-knocked-down cells had elongated mitochondria and abnormal nuclear morphologies (Fig. 6C,D, arrow). Some of these abnormal nuclei showed a structure similar to the apoptotic body (Fig. 6D, arrowhead). However, we could not detect significant differences in the apoptotic level between the control and ObgH1-knocked-down cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (data not shown). It is interesting that the ObgH1-knocked-down cells with an abnormal nuclear morphology still possessed normal nucleolar morphology judging from the immunostaining of nucleolar marker proteins (Fig. 6F).


Figure 6
View larger version (61K):
[in this window]
[in a new window]
  		Figure 6  Knock-down of ObgH1 induced elongated mitochondria and abnormal nuclear morphology. (A) ObgH1-specific siRNA was introduced into HeLa cells (ObgH1-RNAi) and incubated at 37 °C for 2 days. Then total RNA was isolated from the cells and RT-PCR was performed as described in Experimental procedures. The RT-PCR products were subjected to agarose gel electrophoresis, visualized, and quantified. (B) HeLa cells expressing V5-tagged ObgH1 were transfected with siRNA for ObgH1. The total cell lysates from the transfected and untransfected cells were prepared and analyzed by Western blotting using anti-V5 and anti-V5®-actin antibodies. (C, D) The ObgH1-knocked-down cells were stained with Mitotraker Red CMXRos (C) or DAPI and anti-lamin B antibody (D). The arrows in (C) indicate elongated mitochondria. The arrows in (D) show nuclei with abnormal morphologies. An arrowhead shows apoptotic body. Bars: 10 µm. A statistical analysis of the cells with abnormal nucleus was summarized in the right panel in (D). (F) Immunostaining of ObgH1-knocked-down HeLa cells using nucleolar marker protein antibodies as described in Figure 3B.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Evolutionary conservation of Obg function

The present study demonstrated that two human Obg proteins (ObgH1 and ObgH2) have comparable GTPase activities to the other reported Obg subfamily proteins (Table 1 and Fig. 2). Previous studies (Datta et al. 2004), including ours (Sato et al. 2005), have reported that Obg is critical for ribosome maturation. The fact that both ObgH1 and ObgH2 complemented the function of ObgE in E. coli supports the postulated function of these human Obg proteins in ribosome maturation. The immunofluorescence microscopy of HeLa cells revealed that ObgH1 localized in the mitochondria, whereas ObgH2 localized in the nucleolus (Fig. 3). This suggests that ObgH1 and ObgH2 function in the ribosome maturation processes in their respective distinct intracellular organelle.

In the animal cell, the ribosomes are generated in the nucleolus and mitochondria (Curgy 1985; Kitakawa & Isono 1991). In the nucleolus, rRNA is transcribed as a 45S pre-rRNA by RNA polymerase I at the FC/DFC border. Various post-transcriptional modifications, e.g. the 5'- and 3'-processing, and 2'-O-methylation, occur and eventually three rRNAs (28S, 18S, and 5.8 SrRNAs) are generated in the DFC region. These rRNAs and the ribosomal proteins, which are synthesized in the cytoplasm and imported into the nucleus, assemble in the GC region, and are exported back to the cytoplasm. In contrast, the animal mitochondria have a ribosome-organizing mechanism distinct from the nucleolar ribosome biogenesis. The animal mitochondrial genome encodes two rRNA genes (for 16S and 12S, but not 5S rRNA), and the tRNAval gene is inserted in between the 16S and 12S rRNA genes. These genes are transcribed as a single RNA molecule and the rRNA processing occurs at the tRNAval site. Thus, finally, individual 16S and 12S rRNAs are produced. The generated rRNAs assemble with the mitochondrial ribosomal proteins that are encoded by the nuclear genome and translated in the cytoplasm. Our present results indicate that ObgH1 and ObgH2 can function in the different ribosome maturation processes. Taken together with the previous reports (Datta et al. 2004), the small G-proteins, Obg subfamily proteins, in general play a critical role in the fundamental ribosome-maturation process in bacteria and eukaryote.

Specificities of human Obg proteins

Animals and plants have multiple Obg subfamily proteins, although all species in other kingdoms have only one Obg subfamily protein. Our phylogenetic analysis revealed that Obg subfamily proteins cluster into two clades: bacterial and eukaryotic (Fig. 1A). Both of the two human Obg proteins belong to the eukaryotic clade, one for the nucleolus and the other for the mitochondria. ObgH1 was predicted to possess a mitochondria-localization signal at its N-terminus. This type of mitochondria-localization signal has been reported also in S. cerevisiae Obg, Mtg2p, which is the only one member of the S. cerevisiae Obg (Datta et al. 2005). Mtg2p localizes in the mitochondria and participates in ribosome maturation. Other Obg proteins, which are in the eukaryotic clade (except the ObgH2 containing clade), possess the similar primary structure as ObgH1. These facts suggest that almost all eukaryotic Obg proteins localize in the mitochondria and function in the mitochondrial ribosome maturation process.

Our data on the deletion and GFP-fusion of ObgH1 suggest that the amino-terminal region of ObgH1 plays a critical role in the mitochondria localization mechanism/process. However, as seen in Fig. 4C,D, the localization of two mutants (GFP-ObgH1 and ObgH1NC) shows subtle differences. Our interpretation of these results is the following: A complete deletion of the amino-terminal 71 amino acids leads to the nuclear localization; however, GFP, which is fused to the amino terminus, blocks the function as the mitochondria-localization signal, but the rest of the regions could exert a not-yet-identified effect (localization on the nuclear rim).

In contrast, ObgH2 localized in the nucleolus, but it has no significant signal sequence. As mentioned above, no nucleolar localizing Obg protein has been known in S. cerevisiae. On the other hand, plants have two Obg subfamily proteins (Fig. 1A); one (ObgA2) is in the bacterial clade close to cyanobacteria and another one (ObgA1) is in the ObgH1-containing clade. A preliminary experiment in Arabidopsis thaliana showed that ObgA2 localized in the chloroplast and ObgA1 in the mitochondria, i.e. no Obg subfamily protein in A. thaliana functions in the nucleolus (N. Suwastika et al. unpublished observation). These facts support our idea that the nucleolar localizing Obg protein is specific in animals.

In S. cerevisiae, it has been reported that a NOG subfamily protein, Nog1p, which is one of the Obg family proteins, localizes in the nucleolus and participates in ribosome maturation (Jensen et al. 2003). Because, in plant and yeast, no Obg subfamily protein localizes to the nucleolus, the member(s) of the other family (Hflx) and subfamily (NOG, DRG, Ygr210 and YyaF/YchF) is expected to function in the nucleolus.

It is interesting to note that our RNAi knock-down experiment (Fig. 5A) and a previous report (Hernandez-Verdun 2006) showed a reduction of the number of FC region and a breakdown of the nucleolus architecture. Because the reduction of the number of FC region resulted from the inhibition of rDNA transcription (Hernandez-Verdun 2006), ObgH2 might also participate in rDNA transcription. Alternatively, because ObgE forms a dimer and directly binds to rRNA in the GTPase activity-dependent manner (Sato et al. 2005), it might be the case that ObgH2 binds to rRNA and acts as a bridge between RNAs and nucleolus structures.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Database search and phylogenetic analysis

For the 12 eukaryotes, 20 bacteria and 9 archaea whose genome projects had been completed, FASTA search of the genes encoding Obg was conducted on the Kyoto Encyclopedia of Genes and Genomes database (Kanehisa & Goto 2000) using the amino acid sequences for Obg of E. coli and Obg of B. subtilis as queries. The amino acid sequences of the Obg subfamily proteins obtained by FASTA search were aligned with CLUSTALX program (Jeanmougin et al. 1998), and the alignment was used for phylogenetic analysis with the PROTDIST and NEIGHBOR programs of the PHYLIP 3.6 package (Retief 2000). The phylogenetic tree was inferred by neighbor-joining method (Saitou & Nei 1987) and tested by 100 replications of the bootstrap analysis, which was carried out with SEQBOOT and CONSENSE programs in the same package, and then visualized using the TREEVIEW program (Page 1996).

Materials

HeLa S3 cells were cultured in complete Dulbecco's Modified Eagle's Medium (SIGMA, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT, USA) in 5% CO2 at 37 °C. Anti-ObgE antibody and ObgE-deleted E. coli strain have been reported in the previous papers (Kobayashi et al. 2001; Sato et al. 2005). In both immunoblot and immunofluorescence experiments, the anti-ObgE antibody reacts with ObgH2, but not with ObgH1. Anti-fibrillarin monoclonal antibody, anti-B23 polyclonal antibody, anti-actin antibody and anti-V5 antibody were purchased from Cytoskeleton, Inc. (Denver, CO, USA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), SIGMA, and Invitrogen (Carlsbad, CA, USA), respectively. Alexa Fluor 568-conjugated anti-goat IgG, MitoTracker Red CMXRos, and 4',6-diamino-2-phenylindole (DAPI) were purchased form Molecular Probes (Eugene, OR, USA). Rhodamine- or fluorescein isothiocyanate-conjugated anti-mouse IgG and fluorescein isothiocyanate-conjugated anti-rabbit IgG were purchased from Cappel (Irvine, CA, USA). GTP and other regents were purchased from SIGMA and Nacalai Tesque (Kyoto, Japan), respectively.

DNA constructs and immunofluorescence microscopy

The cDNAs for ObgH1 (NCBI gene no. 26164, IMAGE clone ID MGC29512) and ObgH2 (NCBI gene No 85865, FLJ clone ID 38242) were purchased from Invitrogen and Kazusa DNA Research Institute (Kisarazu, Japan), respectively. The full-length ObgH1 and ObgH2 cDNAs were subcloned into pEGFP-C1, pEGFP-N1 (Clontech, Mountain View, CA, USA), pGEX 5X-3 (Amersham Biosciences, Piscataway, NJ, USA), and pcDNA3.2/V5-DEST (Invitrogen). The cDNA fragments corresponding to 1–71 amino acids, 14–406 amino acids, 31–406 amino acids, 51–406 amino acids, and 72–406 amino acids of ObgH1 were amplified by PCR and subcloned into pEGFP-N1 vector. The transfection of HeLa cells were performed using Effectene Transfection regent (Qiagen, Valencia, CA, USA). The microscopic observations were performed as described in the previous report (Yoshimura et al. 2003). All microscopic observation was performed with confocal microscopy except DAPI staining.

Isolation of mitochondria and immunoblotting

Mitochondria were isolated from HeLa cells using Mitochondria Isolation Kit (PIERCE, Rockford, IL, USA) according to the manufacturer's protocol. The isolated sample was subjected to immunoblot analysis using anti-V5 antibody. The purity of the mitochondria fraction was ensured by Western blot analysis using anti-cytochrome c (A8) antibody (Santa Cruz Biotechnology, Inc.), and anti-GAPDH antibody (IMGENEX, San Diego, CA, USA).

GTPase assay

GST-fused full-length ObgH1 and ObgH2 were expressed in E. coli and affinity purified by glutathione-Sepharose beads (Amersham Biosciences). The reaction mix containing 0.4 µM GST-ObgH1 or 2 µM GST-ObgH2, 1 mM GTP and GTPase assay buffer (100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, and 0.1% Tween-20) was incubated at 37 °C for 12 h. The released phosphate was quantified with Biomol green regent (Biomol Research Laboratories, Plymouth Meeting, PA, USA) according to the manufacturer's protocol.

ObgE complementation assay

The plasmid harboring ObgH1 or ObgH2 ORF (pTrc99A amp PtrcobgH1 or obgH2) was introduced into ObgE-deleted E. coli strain (MG1655{Delta}ObgE::kan [ori-pSC101 cat repts PBADobgE+]). Both pTrc99A and the plasmid harboring ObgE are compatible in the E. coli cell. The cells were grown on a LB plate containing ampicillin (100 µg/mL), chloramphenicol (25 µg/mL), and arabinose (0.05%). A single colony was resuspended in phosphate buffer saline, diluted and replated on a LB agar plate containing 0.05% arabinose. The number of colony was counted to determine the number of the cells in the cell suspension. The diluted cells (3 x 103, 3 x 102, 3 x 101 cells for ObgH1 and 9 x 102, 9 x 101, 9 cells for ObgH2, respectively) were spotted onto a LB plate containing antibiotics in the presence or absence of 0.05% arabinose, and 0.01 mM isopropylthiogalactoside and incubated at 30 or 37 °C for 3 days.

RT-PCR

Total RNA was purified from HeLa cells using RNeasy Midi kit (Qiagen). The first strand synthesis was performed using Super script first-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer's protocol. Using this cDNA as a template, full-length cDNAs of ObgH1 and ObgH2 were amplified by PCR.

RNA interference

RNA interference siRNAs for ObgH1 and ObgH2 were designed and synthesized by Invitrogen (Stealth RNAi). The nucleotide sequences of siRNAs were as follows: 5'-UUGGGCUUCAGGGAGGUCAAUCUUG-3' for ObgH1 and 5'-UAAAUGCGUAAUCUGCAAUUGCAGG-3' for ObgH2. Transfection of the HeLa cell was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Two days after the transfection, the cells were subjected to the immunoblotting and immunofluorescence microscopy using anti-V5, ObgH2, ß-actin, fibrillarin, B23, and 2-30C antibodies. 2-30C is a monoclonal antibody that recognizes FC-localizing antigen (Kumeta et al. unpublished observation). For the confirmation of the ObgH1 knock-down, the amount of endogenous mRNA for ObgH1 was determined by RT-PCR. Total RNA was isolated from the control and ObgH1-knocked-down HeLa cells as indicated before. A cDNA pool was generated from 1 µg total RNA and a full-length ObgH1 cDNA was amplified from this cDNA pool by PCR. For an internal control, a full-length GADPH cDNA was also amplified from of the same cDNA pool. These PCR products were subjected to 0.75% agarose gel electrophoresis, visualized by ethidium bromide and quantified using IMAGEQUANT 5.0 software.


   Acknowledgements
 
This study was supported by the Grants-in-Aid for Scientific Research on Priority Areas (for K. T. and S. H. Y.) and COE Research (for K. T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Y. H. is a recipient of the JSPS (Japan Society for the Promotion of Science) predoctoral fellowship. We thank A. Sato for ObgH1 construction, M. Denawa for discussion of our bioinformatic analysis and S. Iwasaka for technical support.


   Footnotes
 
Communicated by: Hiroji Aiba

* Correspondence: E-mail: hirano{at}lif.kyoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bourne, H.R., Sanders, D.A. & McCormick, F. (1991) The GTPase superfamily: conserved structure and molecular mechanism. Nature 349, 117–127.[CrossRef][Medline]

Buglino, J., Shen, V., Hakimian, P. & Lima, C.D. (2002) Structural and biochemical analysis of the Obg GTP binding protein. Structure 10, 1581–1592.[Medline]

Curgy, J.J. (1985) The mitoribosomes. Biol. Cell. 54, 1–38.[Medline]

Datta, K., Skidmore, J.M., Pu, K. & Maddock, J.R. (2004) The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels. Mol. Microbiol. 54, 1379–1392.[CrossRef][Medline]

Datta, K., Fuentes, J.L. & Maddock, J.R. (2005) The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2. Mol. Biol. Cell 16, 954–963.[Abstract/Free Full Text]

Hernandez-Verdun, D. (2006) Nucleolus: from structure to dynamics. Histochem. Cell Biol. 125, 127–137.[CrossRef][Medline]

Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G. & Gibson, T.J. (1998) Multiple sequence alignment with CLUSTAL X. Trends Biochem. Sci. 23, 403–405.[CrossRef][Medline]

Jensen, B.C., Wang, Q., Kifer, C.T. & Parsons, M. (2003) The NOG1 GTP-binding protein is required for biogenesis of the 60 S ribosomal subunit. J. Biol. Chem. 278, 32204–32211.[Abstract/Free Full Text]

Kanehisa, M. & Goto, S. (2000) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30.[Abstract/Free Full Text]

Kitakawa, M. & Isono, K. (1991) The mitochondrial ribosomes. Biochimie 73, 813–825.[Medline]

Kobayashi, G., Moriya, S. & Wada, C. (2001) Deficiency of essential GTP-binding protein ObgE in Escherichia coli inhibits chromosome partition. Mol. Microbiol. 41, 1037–1051.[CrossRef][Medline]

Leipe, D.D., Wolf, Y.I., Koonin, E.V. & Aravind, L. (2002) Classification and evolution of P-loop GTPases and related ATPases. J. Mol. Biol. 317, 41–72.[CrossRef][Medline]

Lin, B., Covalle, K.L. & Maddock, J.R. (1999) The Caulobacter crescentus CgtA protein displays unusual guanine nucleotide binding and exchange properties. J. Bacteriol. 181, 5825–5832.[Abstract/Free Full Text]

Lin, B., Skidmore, J.M., Bhatt, A., Pfeffer, S.M., Pawloski, L. & Maddock, J.R. (2001) Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function. Mol. Microbiol. 39, 924–934.[CrossRef][Medline]

Lin, B., Thayer, D.A. & Maddock, J.R. (2004) The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit. J. Bacteriol. 186, 481–489.[Abstract/Free Full Text]

Morimoto, T., Loh, P.C., Hirai, T., Asai, K., Kobayashi, K., Moriya, S. & Ogasawara, N. (2002) Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis. Microbiology 148, 3539–3552.[Abstract/Free Full Text]

Okamoto, S. & Ochi, K. (1998) An essential GTP-binding protein functions as a regulator for differentiation in Streptomyces coelicolor. Mol. Microbiol. 30, 107–119.[CrossRef][Medline]

Page, R.D. (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357–358.[Free Full Text]

Retief, J.D. (2000) Phylogenetic analysis using PHYLIP. Methods Mol. Biol. 132, 243–258.[Medline]

Saitou, N. & Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.[Abstract]

Sato, A., Kobayashi, G., Hayashi, H., Yoshida, H., Wada, A., Maeda, M., Hiraga, S., Takeyasu, K. & Wada, C. (2005) The GTP binding protein Obg homolog ObgE is involved in ribosome maturation. Genes Cells 10, 393–408.[Abstract/Free Full Text]

Scott, J.M. & Haldenwang, W.G. (1999) Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma (B). J. Bacteriol. 181, 4653–4660.[Abstract/Free Full Text]

Scott, J.M., Ju, J., Mitchell, T. & Haldenwang, W.G. (2000) The Bacillus subtilis GTP binding protein Obg and regulators of the sigma (B) stress response transcription factor cofractionate with ribosomes. J. Bacteriol. 182, 2771–2777.[Abstract/Free Full Text]

Sikora, A.E., Zielke, R., Datta, K. & Maddock, J.R. (2006) The Vibrio harveyi GTPase CgtAV is essential and is associated with the 50S ribosomal subunit. J. Bacteriol. 188, 1205–1210.[Abstract/Free Full Text]

Slominska, M., Konopa, G., Wegrzyn, G. & Czyz, A. (2002) Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein. Biochem. J. 362, 579–584.[CrossRef][Medline]

Tan, J., Jakob, U. & Bardwell, J.C. (2002) Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase. J. Bacteriol. 184, 2692–2698.[Abstract/Free Full Text]

Welsh, K.M., Trach, K.A., Folger, C. & Hoch, J.A. (1994) Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis. J. Bacteriol. 176, 7161–7168.[Abstract/Free Full Text]

Wout, P., Pu, K., Sullivan, S.M., Reese, V., Zhou, S., Lin, B. & Maddock, J.R. (2004) The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase. J. Bacteriol. 186, 5249–5257.[Abstract/Free Full Text]

Yoshimura, S.H., Kim, J. & Takeyasu, K. (2003) On-substrate lysis treatment combined with scanning probe microscopy revealed chromosome structures in eukaryotes and prokaryotes. J. Electron Microsc. (Tokyo) 52, 415–423.[Abstract/Free Full Text]

Zhang, S. & Haldenwang, W.G. (2004) Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes. Biochem. Biophys. Res. Commun. 322, 565–569.[CrossRef][Medline]

Received: 15 May 2006
Accepted: 17 August 2006




This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
Y. R. Lapik, J. M. Misra, L. F. Lau, and D. G. Pestov
Restricting Conformational Flexibility of the Switch II Region Creates a Dominant-Inhibitory Phenotype in Obg GTPase Nog1
Mol. Cell. Biol., November 1, 2007; 27(21): 7735 - 7744.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirano, Y.
Right arrow Articles by Takeyasu, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirano, Y.
Right arrow Articles by Takeyasu, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS