Assembly of the Trypanosoma brucei 60S Ribosomal Subunit Nuclear Export Complex Requires Trypanosome-Specific Proteins P34 and P37

Cell culture.Procyclic-form T. brucei brucei strain 427 was grown in Cunningham's medium supplemented with 10% fetal calf serum (6). Clonal P34/P37 RNAi cells have been described previously (19). Induction of double-stranded RNA specific to P34 and P37 was initiated by addition of tetracycline (1 μg/ml; Sigma, St. Louis, MO) to a concentration of 1 × 10⁶ cells/ml. Cells were harvested 4 days postinduction and Western blot analysis was performed, probing for P34 and P37 to confirm the absence of both proteins.

Antibodies.The antibodies used for this study were as follows: P34/P37 polyclonal antiserum (47), affinity-purified P34/P37 polyclonal antibody (Bethyl Laboratories, Montgomery, TX), L3 polyclonal antiserum (Affinity Bioreagents, Golden, CO), L11 polyclonal antiserum (Affinity Bioreagents), L10 monoclonal antibody (Novus Biologicals, Littleton, CO), affinity-purified L5 polyclonal antibody (Bethyl Laboratories), S6 polyclonal antiserum (Affinity Bioreagents), affinity-purified Nmd3 polyclonal antiserum (a gift from Arlen Johnson), affinity-purified T. brucei exportin 1 polyclonal antibody (Bethyl Laboratories), NOG1 and phosphoglycerate kinase polyclonal antibodies (gifts from Marilyn Parsons), and TATA binding protein polyclonal antiserum (a gift from Vivian Bellofatto).

Specificity of antibodies.The ribosomal protein antibodies used in this study were commercially obtained and were raised to recombinant human proteins. Prior to testing the specificity of these antibodies to the trypanosome proteins, we aligned each of the human protein sequences with those from T. brucei (see Fig. S1 to S5 in the supplemental material). We next performed immune capture experiments (described below) with cytoplasmic and nuclear extracts specific for L3, L11, L10, S5, and S6. Subsequent Western blot analyses using the same antibodies used for the immune capture showed that each antibody demonstrated reactivity to the appropriately sized protein and that the majority of each protein was present in the pellet fraction compared to the supernatant (see Fig. S6 in the supplemental material).

We also aligned the yeast Nmd3 protein sequence with the homologous T. brucei protein, which revealed a high degree of identity between the two species (see Fig. S7 in the supplemental material). We utilized an antibody raised to yeast Nmd3 (a generous gift from Arlen Johnson) for Western blot analyses on nuclear and cytoplasmic extracts, the results of which demonstrated that this antibody cross-reacts with a protein of the appropriate molecular weight (data not shown). These results confirmed that T. brucei contains an Nmd3 homologue and that the yeast antibody was useful for further experiments.

Cellular fractionation.Cytoplasmic and whole nuclear extracts were prepared as described previously (19). Nucleolar and nucleoplasmic extracts were prepared as described previously (36).

Immune capture experiments.Immune capture experiments were done as described previously (19) using antibodies specific to the following proteins: P34 and P37, exportin 1, and L3, L11, L10, S5, and S6. Briefly, 500 μg of the indicated extract was incubated with antibody-cross-linked Dynabeads (Invitrogen, Carlsbad, CA). After incubation, the beads were washed with phosphate-buffered saline and eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. For each immune capture experiment, the protein(s) present in the pellet fractions represents the majority of the protein, compared to that in the unbound fraction (data not shown).

Western blot analyses.Following SDS-PAGE, the gels were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) and blocked in 10% nonfat dry milk. Membranes were incubated with the indicated primary antibody overnight, followed by 10 washes with Tris-buffered saline with Tween and incubation with the appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were detected using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).

RT-PCR analyses.Immune capture experiments were performed as described above, with the modification of incubating the Dynabeads with poly(dI-dC) (Sigma, St. Louis, MO) overnight in order to decrease nonspecific RNA binding. The beads were eluted by resuspension in elution buffer (1% SDS, 50 mM dithiothreitol, and 10% β-mercaptoethanol) and boiling for 5 min. Bound RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. Subsequent reverse transcription-PCR (RT-PCR) analyses were performed using primers specific to internal sequences of processed 60S rRNA (28Sβ), 40S rRNA (18S), and the entire processed 5S rRNA sequence (5). A fraction of each reaction mixture was subjected to agarose gel electrophoresis and ethidium bromide staining to visualize the RT-PCR products.

Sequential immune capture experiments.Sequential immune capture experiments were performed as described previously (19). Briefly, the primary immune capture against exportin 1 was performed as described for the single immune capture experiments with the modification of starting with 2 mg of nuclear extract. The beads were eluted as described for the RT-PCR analyses, followed by dilution with 1 ml of nondenaturing lysis buffer. Samples were allowed to cool to room temperature and were then used for a secondary immune capture experiment specific for L11. P34/P37 Western blot analyses were performed on the resulting pellet fractions.

Subcellular fractionation demonstrates that P34 and P37 are present in the nucleolar fraction of procyclic cells.Previous work in our laboratory has demonstrated that P34 and P37 are present in the 80S fractions of polysomal preparations (18) and, more recently, we have shown that they are also present in the 60S and polysomal fractions (M. Ciganda, unpublished data). We next wished to determine more specifically when and where these proteins become associated with the 60S ribosomal subunit. Subcellular fractionation was employed in order to obtain wild-type procyclic whole nuclear, nucleoplasmic, nucleolar, and cytoplasmic extracts. It should be noted that the whole nuclear extract contains both nucleoplasmic and nucleolar material, which is further fractionated to yield the separate nucleoplasmic and nucleolar extracts. Western blot analyses probing for cellular compartmental markers for each fraction demonstrated that we had isolated distinct subcellular fractions (Fig. 2B), which could be used in subsequent immune capture experiments. In addition, Western blot analysis probing for P34 and P37 demonstrated that both proteins were present in each of our subcellular fractions (Fig. 2A). This figure is representative of three separate experiments. The presence of these proteins in the nucleolar fraction, which had not been demonstrated previously, suggests that they may be involved in earlier nucleolar stages of ribosomal biogenesis. Thus, utilizing subcellular fractionation in conjunction with immune capture experiments, we sought to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. Since we had determined previously that P34 and P37 associate with both 5S rRNA (33) and ribosomal protein L5 (Hellman et al., unpublished), two components of the 60S ribosomal subunit, we focused our initial experiments on potential 60S ribosomal protein binding partners.

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FIG. 2.

P34 and P37 are present in the nucleolar fraction of procyclic cells. Wild-type procyclic cells were fractionated into whole nuclear, nucleolar, nucleoplasmic, and cytoplasmic extracts. (A) A fraction of each extract was subjected to SDS-PAGE and Western blot analysis probing for P34 and P37. (B) A fraction of each extract was subjected to SDS-PAGE and Western blot analysis probing for specific cellular compartmental markers. NOG1, a nucleolar marker; TBP (TATA binding protein), a nucleoplasmic marker; PGK (phosphoglycerate kinase), a cytoplasmic marker. CE, 50 μg of cytoplasmic extract; NE, 50 μg of whole nuclear extract; NO, 100 μg of nucleolar extract; NP, 100 μg of nucleoplasmic extract.

P34 and P37 associate with L3-containing 60S ribosomal subunits.One of the earliest ribosome-associating 60S proteins is L3, which is added to the 90S particle in the nucleolus (27, 35). The association of a protein(s) with L3, particularly in the nucleolus, may be indicative that it functions early in ribosomal biogenesis. Immune capture experiments specific for L3 were performed with whole nuclear, nucleolar, nucleoplasmic, and cytoplasmic extracts from T. brucei, followed by Western blot analyses probing for P34 and P37. These experiments demonstrated that P34 and P37 associated with L3, either directly or indirectly, in all fractions tested (Fig. 3A, P lanes). Although P37 was not detected in the immune capture pellet fraction for all extracts, additional Western blot analyses demonstrated that it is present in each of these fractions (data not shown) but may not be seen in all blots due to its lower abundance relative to P34 (47). In addition, we confirmed that our antibody precipitated L3 in each fraction by performing L3 Western blot analyses on each pellet fraction (data not shown). These experiments demonstrated that P34 and P37 are associated with the maturing 60S ribosomal subunit throughout the biogenesis pathway, beginning at an early, nucleolar step.

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FIG. 3.

P34 and P37 associate with 60S protein L3. Immune capture experiments specific to L3 were performed with whole nuclear, nucleolar, nucleoplasmic, and cytoplasmic extracts. (A) Western blot analyses were performed, probing for P34 and P37. B, immune capture beads alone; NE, 50 μg of whole nuclear extract; NO, 50 μg of nucleolar extract; NP, 50 μg of nucleoplasmic extract; CE, 50 μg of cytoplasmic extract; P, immune capture pellet fraction. (B) Diagram depicting the ribosomal complexes and rRNA species with which L3 associates. (C) RT-PCR analyses performed on RNA extracted from L3 immune capture experiments using cytoplasmic and whole nuclear extracts. 1 kb, 1-kb DNA ladder (Invitrogen); -RT, no-RT control; 28S, 28S RT-PCR; 18S, 18S RT-PCR; 5S, 5S RT-PCR; B, immune capture beads alone (control); CE, cytoplasmic extract immune capture; NE, whole nuclear extract immune capture. Results shown are representative of at least three separate experiments.

In order to ensure that L3 was being precipitated in the context of the 60S subunit and not simply free protein, we performed RT-PCR analyses on immune capture pellet fractions from experiments performed with cytoplasmic and nuclear extracts. RT-PCR experiments were performed specific to 60S rRNA (28S and 5S) and 40S rRNA (18S) species.

The rRNA species found in association with L3, as demonstrated in yeast (27), are shown in Fig. 3B. L3 associates with the early 90S particle in yeast (Fig. 1), and if this is also true for T. brucei, it should be complexed with not only 28S and 5S rRNA in nuclear extracts but also 18S rRNA. In addition, L3 should associate with 28S and 5S rRNA in cytoplasmic extracts, since it remains associated with mature 60S subunits, and with 18S rRNA subsequent to subunit joining. RT-PCR analyses performed on RNA extracted from nuclear and cytoplasmic L3 immune capture experiments from T. brucei demonstrated that L3 does associate with 28S, 18S, and 5S rRNA in both subcellular fractions (Fig. 3C). These results demonstrate that L3 in our immune capture experiments is precipitating 60S complexes, although we cannot rule out the possibility that some of the L3 is free protein.

P34 and P37 associate with 40S ribosomal protein S5-containing ribosomal particles in nucleolar and whole nuclear extracts.An association of P34 and P37 with L3-containing 60S particles in the nucleolar fraction may be interpreted in one of two ways: (i) P34 and P37 are associated with 90S particles or (ii) P34 and P37 associate with the subunit subsequent to the rRNA cleavage event that gives rise to the 66S and 43S subunits. Both 90S and 66S particles are present in the nucleolus and, thus, it is important to distinguish between them to more precisely determine the 60S particle(s) with which P34 and P37 associate. In order to determine whether P34 and P37 are involved in ribosomal biogenesis at the initial 90S particle, we performed immune capture experiments specific to S5, a 40S ribosomal subunit protein that comes into the pathway at the 90S particle stage (Fig. 1) (15). Since P34 and P37 have thus far been shown to associate only with specific 60S subunit factors, an association with S5 in whole nuclear or nucleolar extracts would be indicative of P34 and P37 being present in 90S particles. Immune capture experiments specific to S5 followed by Western blot analyses probing for P34 and P37 demonstrated that both proteins are complexed with S5 in whole nuclear and nucleolar extracts (Fig. 4A, P lanes), which indicates that these proteins become involved in 60S subunit biogenesis at the 90S nucleolar stage prior to subunit separation. However, neither protein associates with S5-containing particles in the nucleoplasm (Fig. 4A, P lane), demonstrating that the association between P34 and P37 and S5 is specific to the precleaved particle and, subsequent to cleavage, they are found specifically with the 60S subunit. In addition, RT-PCR experiments performed on P34/P37 immune captures using whole nuclear extracts demonstrate a complex between these proteins and 18S rRNA, most likely while it is still part of the 35S rRNA precursor (data not shown).

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FIG. 4.

P34 and P37 associate with S5 in whole nuclear and nucleolar extracts. Wild-type procyclic whole nuclear, nucleolar, and nucleoplasmic extracts were used for immune capture experiments specific for 40S protein S5. (A) Western blot analyses were performed, probing for P34 and P37. B, immune capture beads alone; NE, 50 μg of whole nuclear extract; NO, 50 μg of nucleolar extract; NP, 50 μg of nucleoplasmic extract; P, immune capture pellet fraction. (B) Diagram depicting the ribosomal complexes and rRNA species with which S5 associates. (C) RT-PCR analyses performed on RNA extracted from S5 immune capture experiments using cytoplasmic and whole nuclear extracts. 1 kb, 1-kb DNA ladder (Invitrogen); -RT, no-RT control; 28S, 28S RT-PCR; 18S, 18S RT-PCR; 5S, 5S RT-PCR; B, immune capture beads alone (control); CE, cytoplasmic extract immune capture; NE, whole nuclear extract immune capture. Results shown are representative of at least three separate experiments.

The rRNA species with which S5 associates in yeast are demonstrated in Fig. 4B. Since S5 is a 40S protein that is added to the 90S particle in the nucleolus and remains associated throughout the 40S subunit maturation pathway (15, 27), it should associate with 18S and 28S rRNA species in nuclear extracts and all three rRNA species in cytoplasmic extracts (Fig. 4B). Our results demonstrate that S5 is complexed with 28S, 18S, and 5S rRNA in both nuclear and cytoplasmic extracts (Fig. 4C). There is some controversy over the stage at which 5S rRNA becomes incorporated into the yeast 60S subunit. Some studies have indicated that it integrates into 90S particles (46), whereas others suggest that it is incorporated into the 66S particle (7). Our results demonstrate that, in T. brucei, 5S rRNA incorporates into the 90S ribosomal particle.

P34 and P37 associate with L11-containing 60S ribosomal subunits.In order to confirm that P34 and P37 remain complexed with the developing 60S ribosomal subunit, we used L11 as a marker for the late-associating proteins that are part of the 66S ribosomal particle (Fig. 1). Immune capture experiments specific for L11 demonstrated an association between P34 and P37 and L11, which is either direct or indirect, in all extracts tested (Fig. 5A, P lanes). Western blot analyses probing for L11 demonstrated that this protein was precipitated from each extract (data not shown).

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FIG. 5.

P34 and P37 associate with L11. Wild-type procyclic nuclear, nucleolar, nucleoplasmic, and cytoplasmic extracts were used for immune capture experiments specific for L11. (A) Western blot analyses were performed, probing for P34 and P37. B, immune capture beads alone; NE, 50 μg of whole nuclear extract; NO, 50 μg of nucleolar extract; NP, 50 μg of nucleoplasmic extract; CE, 50 μg of cytoplasmic extract; P, immune capture pellet fraction. (B) Diagram depicting the ribosomal complexes and rRNA species with which L11 associates. (C) RT-PCR analyses performed on RNA extracted from L11 immune capture experiments using cytoplasmic and whole nuclear extracts. 1 kb, 1-kb DNA ladder (Invitrogen); -RT, no-RT control; 28S, 28S RT-PCR; 18S, 18S RT-PCR; 5S, 5S RT-PCR; B, immune capture beads alone (control); CE, cytoplasmic extract immune capture; NE, whole nuclear extract immune capture. Results shown are representative of at least three separate experiments.

Figure 5B shows the rRNA species associated with L11 throughout the yeast 60S subunit biogenesis pathway. Because L11 assembles into the maturing 60S particle at the 66S stage (Fig. 1 and 5B) (16), it should be complexed with 28S and 5S rRNA, but not 18S, in T. brucei nuclear extracts if this follows the yeast model. In cytoplasmic extracts, L11 should associate with 28S and 5S rRNA through its involvement with the mature 60S subunit and 18S rRNA in the context of the 80S ribosome (Fig. 5B). RT-PCR analyses performed on L11 immune capture experiments from T. brucei demonstrate that it associates with 28S and 5S rRNA but not 18S rRNA (Fig. 5C) in the nucleus and with all three rRNA species in cytoplasmic extracts (Fig. 5C).

The results shown in Fig. 5 demonstrate that P34 and P37 associate with the developing 60S ribosomal subunit at multiple steps during its biogenesis, an unusual characteristic for trans-acting factors involved in ribosomal biogenesis. The trans-acting factors typically act on one or two ribosomal particle intermediates, after which they are released from the particle and replaced with subsequent factors (37).

In addition, since P34 and P37 were found to associate with 60S particles in both nuclear and cytoplasmic fractions, we next sought to determine whether they are components of the 60S nuclear export complex. Previous results demonstrated an association between P34 and P37 and exportin 1 (19), which is one of the factors required for the transport of nuclear pre-60S subunits to the cytoplasm in yeast and mammalian cells (14, 20, 40). We then hypothesized that this P34/P37-exportin 1 association may occur in the context of the 60S ribosomal subunit. Although the 60S nuclear export complex has been elucidated in other organisms and found to contain exportin 1 and Nmd3, a nuclear export signal (NES)-containing adaptor protein (14, 20, 41), this has not been studied in T. brucei. Therefore, we first needed to determine whether T. brucei utilizes a nuclear export complex similar to that described for yeast and mammals.

T. brucei 60S subunits associate with exportin 1.In order to determine whether T. brucei 60S ribosomal subunits associate with exportin 1, we performed immune capture experiments specific for L3 and L11 using whole nuclear extracts. These experiments demonstrated that L3- and L11-containing 60S particles associate with exportin 1, although each does so to a different extent (Fig. 6A, P lanes). We do not currently know if this is due to stoichiometric differences or if it is due to our experimental procedures (e.g., differences in the efficiencies of the antibodies used in the immune capture). These results demonstrate that T. brucei 60S subunits form an exportin 1-containing nuclear export complex.

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FIG. 6.

T. brucei 60S ribosomal proteins associate with exportin 1 and Nmd3. Immune capture experiments specific to 60S proteins L3 and L11 were performed using wild-type whole nuclear extracts. Subsequent Western blot analyses were performed probing for exportin 1 (A) and Nmd3 (B). B, immune capture beads alone; NE, 50 μg of whole nuclear extract; P, immune capture pellet fraction. Results shown are representative of at least three separate experiments.

T. brucei 60S subunits associate with an Nmd3 homologue.In addition to exportin 1, it has been shown that Nmd3 is required for the nuclear export of 60S ribosomal subunits by providing the NES for the 60S-exportin 1 interaction (14, 20, 41). However, it was not known whether T. brucei contains an Nmd3 protein homologue, so we used the yeast Nmd3 sequence to query the T. brucei database and found a homologous protein (accession number XP_845721). The two sequences were aligned using ClustalW, which found an approximately 40% identity between the protein sequences from both organisms (see Fig. S7 in the supplemental material). We also determined that an Nmd3 antibody which had been raised to the yeast protein cross-reacted with an appropriately sized protein in T. brucei extracts (data not shown).

Since we had determined that T. brucei contains an Nmd3 homologue, we next wished to determine whether exportin 1 and 60S subunits form a nuclear export complex containing this protein. Immune capture experiments specific to exportin 1, L3, and L11 were performed using wild-type procyclic nuclear extracts, followed by Western blot analyses probing for Nmd3. Each protein showed an interaction with Nmd3 (Fig. 6B, P lanes), demonstrating that the T. brucei 60S ribosomal subunit forms a nuclear export complex containing Nmd3.

P34 and P37 associate with the 60S nuclear export complex.Having now demonstrated that T. brucei 60S ribosomal subunits form a complex with both exportin 1 and Nmd3, we next wished to determine whether P34 and P37 are a part of this complex. We have shown previously that P34 and P37 associate with exportin 1 (19). In order to determine whether this complex also includes pre-60S subunits that are loaded with Nmd3, we performed immune capture experiments specific for P34 and P37 using procyclic whole nuclear extracts. Subsequent Western blot analyses probing for Nmd3 demonstrated that P34 and P37 associate with Nmd3 (Fig. 7A).

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FIG. 7.

P34 and P37 associate with the 60S nuclear export complex. (A) Wild-type whole nuclear extract was used for immune capture experiments specific for P34 and P37. Subsequent Western blot analysis was performed, probing for Nmd3. (B) Wild-type whole nuclear extract was used for sequential immune capture experiments. The first capture was performed specific to exportin 1 (XpoI), which was eluted and used for a second capture specific for L11. Subsequent Western blot analyses were performed probing for P34 and P37. B, immune capture beads alone; NE, 25 μg of whole nuclear extract; P, sequential immune capture pellet fraction; P2, sequential immune capture second elution. Results shown are representative of at least three separate experiments.

In order to confirm that P34 and P37 associate with exportin 1 and the 60S subunit simultaneously, we performed sequential immune capture experiments using nuclear extracts. We have used this technique previously to establish the complex formed between P34, P37, NOPP44/46, and exportin 1 (19). Performing a series of immune captures allows for enrichment of complexes containing specific proteins. We performed the first immune capture specific to exportin 1, which was eluted, renatured, and utilized for a second capture specific to L11. Western blot analyses probing for P34 and P37 demonstrated that these proteins are present in a complex containing both exportin 1 and 60S, although P37 may have been below the level of detection in these experiments due to its lower expression relative to P34 (Fig. 7B). This confirms that P34 and P37 are components of the 60S nuclear export complex and do not simply facilitate the association between 60S and exportin 1.

T. brucei 60S ribosomal subunits fail to assemble with the nuclear export machinery in the absence of P34 and P37.As P34 and P37 are trypanosome-specific proteins associated with the 60S nuclear export complex, which is formed in organisms that lack these proteins, we hypothesized that P34 and P37 may be required for formation of this complex in T. brucei. Utilizing a previously constructed P34/P37 RNAi cell line (18), we examined the 60S-Nmd3 and 60S-exportin 1 interactions in the absence of P34 and P37.

P34/P37 RNAi cells were grown to the appropriate density (1 × 10⁶ cells/ml), at which point tetracycline (1 μg/ml) was used to induce double-stranded RNA expression specific for P34 and P37. The cells were harvested and fractionated into nuclear and cytoplasmic extracts at 4 days postinduction, and Western blot analysis probing for P34 and P37 confirmed the absence of both proteins in each extract (data not shown). It is important to note that the cells were harvested at a point where P34 and P37 were no longer detectable by Western blot analysis but the cells were still viable.

In order to determine whether the association between 60S subunits and Nmd3 was altered in the P34/P37 RNAi cells, we repeated our L3 and L11 immune capture experiments using the nuclear extracts prepared from these cells. These experiments were performed in tandem with the wild-type nuclear extracts in order to make a direct comparison of the 60S-Nmd3 association between the two cell lines. Nmd3 Western blot analyses for the L3 and L11 immune capture experiments performed with wild-type and P34/P37 RNAi nuclear extracts demonstrated that neither L3 nor L11 associates with Nmd3 in the absence of P34 and P37 (Fig. 8A, P lanes, compare wild type to RNAi).

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FIG. 8.

P34 and P37 are required for the association between 60S and Nmd3. P34/P37 RNAi whole nuclear extracts were used for L3 and L11 (A) and L10 (B) immune capture experiments, and Western blot analyses were performed probing for Nmd3. B, immune capture beads alone; NE, 50 μg of wild-type whole nuclear extract; P, immune capture pellet fraction; WT, wild type; RNAi, P34/P37 RNAi. Results shown are representative of at least three separate experiments.

As a control, we examined the association between L10 and Nmd3 in P34/P37 RNAi cells. L10 is a 60S protein that associates directly with Nmd3 in the absence of 60S subunits (14). Therefore, loss of P34 and P37 should not alter the association of L10 with Nmd3. Immune capture experiments specific to L10 using wild-type and P34/P37 RNAi nuclear extracts demonstrated that P34 and P37 are not required for the direct protein-protein interaction between L10 and Nmd3 (Fig. 8B, P lanes, compare wild type to RNAi) but are required for the 60S-Nmd3 interaction. These data demonstrate that P34 and P37 mediate the 60S-Nmd3 interaction in the context of the whole subunit rather than individual protein-protein interactions.

It has been shown in both yeast and mammalian cells that Nmd3 is an adaptor protein that mediates the association between the 60S ribosomal subunit and exportin 1 by providing the NES in trans (14, 20). Thus, it was of interest to examine what happens to the association between the 60S subunit and exportin 1 in the absence of P34 and P37, as we have shown in Fig. 8 that the association between 60S and Nmd3 is altered in these cells. We performed the same immune capture experiments as described for Fig. 8, using wild-type and P34/P37 RNAi nuclear extracts. Subsequent Western blot analyses probing for exportin 1 demonstrated that the association between 60S and exportin 1 is lost in the absence of P34 and P37 (Fig. 9, P lanes, compare wild type to RNAi). Together, these results demonstrate that P34 and P37, either directly or indirectly, are required for proper assembly of the 60S ribosomal subunit nuclear export complex in T. brucei.

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FIG. 9.

P34 and/or P37 is required for the association between 60S and exportin 1. Wild-type and P34/P37 RNAi whole nuclear extracts were used for L3 and L11 immune capture experiments. Subsequent Western blot analyses were performed probing for exportin 1. B, immune capture beads alone; NE, 100 μg of wild-type whole nuclear extract; P, immune capture pellet fraction; WT, wild type; RNAi, P34/P37 RNAi. Results shown are representative of at least three separate experiments.

In addition to the experimental results shown, we have performed Western blot analyses specific to L11 using equivalent amounts of wild-type and P34/P37 RNAi nuclear extracts. Results from these experiments demonstrate that L11 accumulates in the nucleus when P34 and P37 are not present (approximately 3.5-fold ± 0.01-fold based on three separate experiments [data not shown]), suggesting a defect in 60S ribosomal subunit nuclear export. Also, preliminary results from two-dimensional difference gel electrophoresis performed with P34/P37 RNAi cytoplasmic extracts have shown that L11 protein levels decrease approximately 2.5-fold in the cytoplasm (L. Wang, unpublished data), lending further support to an alteration in the nuclear export of 60S subunits in the absence of P34 and P37.

P34 and P37 associate with 80S ribosomes.Since we had established that P34 and P37 associate with 60S subunits in the cytoplasm (Fig. 3 and 5), we next wanted to determine whether they remain so subsequent to 60S-40S subunit joining. We did not detect an association between P34 and P37 and S5 in the nucleoplasm (Fig. 6), suggesting that they act specifically on the 60S biogenesis pathway. A complex containing P34, P37, and a 40S protein in the cytoplasm would indicate that this was due to the subunits being joined to form an 80S ribosome. However, since we determined that P34 and P37 are complexed with S5 in whole nuclear and nucleolar extracts, we chose to use a 40S protein that does not associate with the 90S particle as our marker for 80S ribosomes (40S protein S6). Thus, a complex formed between P34 and P37 and S6 in the cytoplasm could only be due to their association with 80S ribosomes.

Thus, in order to determine whether P34 and P37 are complexed with 80S ribosomes, we performed immune capture experiments specific to the 40S protein S6 using cytoplasmic and nuclear extracts. Western blot analyses of the resulting pellets demonstrated that P34 and P37 associate with S6 in cytoplasmic, but not nuclear, extracts (Fig. 10A, P lanes).

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FIG. 10.

P34 and P37 associate with 40S protein S6 in cytoplasmic extracts. Immune capture experiments specific to S6 were performed using wild-type whole nuclear and cytoplasmic extracts. (A) Western blot analyses were performed probing for P34 and P37. B, immune capture beads alone; NE, 50 μg of whole nuclear extract; CE, 50 μg of cytoplasmic extract; P, immune capture pellet fraction. (B) Diagram depicting the ribosomal complexes and rRNA species with which S6 associates. (C) RT-PCR analyses performed on RNA extracted from S6 immune capture experiments using cytoplasmic and whole nuclear extracts. 1 kb, 1-kb DNA ladder (Invitrogen); -RT, no-RT control; 28S, 28S RT-PCR; 18S, 18S RT-PCR; 5S, 5S RT-PCR; B, immune capture beads alone (control); CE, cytoplasmic extract immune capture; NE, whole nuclear extract immune capture. Results shown are representative of at least three separate experiments.

We have illustrated the rRNA species complexed with S6 in yeast in Fig. 10B. S6 incorporates into the maturing yeast 40S subunit in the nucleolus at the 43S particle (Fig. 1 and 10B) (11, 27). If this model is conserved in T. brucei, S6 should associate only with 18S rRNA in nuclear extracts, but it should associate with 28S, 18S, and 5S rRNA in cytoplasmic extracts. Our results demonstrate that these are in fact the rRNA species with which T. brucei S6 associates in each extract (Fig. 10C), demonstrating that we efficiently precipitated 40S ribosomal subunits.

The nuclear results from these experiments revealed that P34 and P37 play a specific role in 60S ribosomal subunit biogenesis, as there is no association with the 40S subunit. The cytoplasmic results demonstrate that P34 and P37 are complexed with 80S ribosomes, suggesting that they remain associated with the translational apparatus, possibly fulfilling a role in subunit joining or translation initiation.