Noreen Williams

ATP Synthase Is Responsible for Maintaining Mitochondrial Membrane Potential in Bloodstream Form Trypanosoma brucei

ABSTRACT The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F1-ATPase α and β subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the β subunit decreased the mRNA and protein levels for the β subunit, as well as the mRNA and protein levels of the α subunit. Similarly, RNAi of α subunit decreased the α subunit transcript and protein levels, as well as the β-subunit transcript and protein levels. In contrast, α and β RNAi knockdown resulted in a 60% increase in the F0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F0-32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for α or β subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of α and β RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.

Eukaryotic 5S rRNA biogenesis.

The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. WIREs RNA 2011 2 523–533 DOI: 10.1002/wrna.74

Developmental regulation of two nuclear RNA binding proteins, p34 and p37, from Trypanosoma brucei

We have previously reported the purification of two closely related nucleic acid binding proteins, p34 and p37, from Trypanosoma brucei and the cloning and sequencing of the two genes encoding these two proteins. The predicted primary structures of the two proteins are nearly identical with one major and several minor differences. Three sequence motifs have been identified in both proteins: an N-terminal alanine, proline, and lysine rich domain, one and a half internal consensus RNA binding domains, and a C-terminal KKDX repeat region. p34 and p37 bind preferentially to heterogeneous RNA as compared with other nucleic acids. Here, we report the developmental regulation of the expression of these two highly related proteins and their intracellular localization in T. brucei. The results indicate that these two RNA binding proteins are differently regulated through the Trypanosoma brucei life cycle. The steady state level of p34 transcript and protein are highest in the procyclic form. In bloodstream form, however, the p34 message is readily detectable, while the protein is not detectable. The p37 transcript level is nearly as high as that for p34 in procyclic form, while the p37 protein level is low. In bloodstream form p37 protein does correlate with the relative abundance of the steady state mRNA level. The two proteins have been localized to the nucleus by immunofluorescent confocal microscopy and subcellular fractionation.

Purification, cloning, and expression of two closely related Trypanosoma brucei nucleic acid binding proteins

Nucleic acid binding proteins in the trypanosomatid family are of particular interest because of several unusual molecular phenomena discovered in these organisms. We have purified two closely related proteins, p34 and p37, from the procyclic from of T. brucei using high salt extraction and single-stranded-DNA (ssDNA) agarose chromatography. Antibodies raised against the p34 protein showed crossreactivity with p37, suggesting relatedness. High performance liquid chromatography analysis and microsequencing of tryptic peptides derived from p34 and p37 showed that the primary structures of the two proteins are nearly identical. We have cloned and sequenced the two genes encoding these two proteins. Protein sequences predicted from the cDNAs confirm the relatedness of the two proteins but also indicate the presence of an 18 amino acid insertion unique to one of the two proteins as well as several minor differences resulting from single amino acid changes. Three sequence motifs have been identified in both proteins: an N-terminal alanine, proline, and lysine rich domain, one and a half internal RNA-binding domains, and a C-terminal KKDX repeat region. Both proteins preferentially bind to heterogenous RNA and ssDNA versus double-stranded DNA and homopolymers. Both recombinant proteins have been expressed in E. coli and show properties indistinguishable from those observed with native p34/p37.

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

ABSTRACT We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.

Proton ATPase of rat liver mitochondria: A rapid procedure for purification of a stable, reconstitutively active F1 preparation using a modified chloroform method

A method is described for the purification of rat liver F1-ATPase by a modification of the chloroform extraction procedure originally described by Beechey et al. (Biochem. J. (1975) 148, 533). Purified liver membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The procedure yields pure F1 in only 2-3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the alpha, beta, gamma, delta, and epsilon bands characteristic of F1-ATPase. It has a high ATPase specific activity, and is reconstitutively active, catalyzing high rates of ATP synthesis. Significantly, it can be readily crystallized. If desired, the enzyme can be passed over a gel filtration column to place it in a stabilizing phosphate-EDTA buffer, lyophilized and stored indefinitely at -20 degrees C.

Two Trypanosome-Specific Proteins Are Essential Factors for 5S rRNA Abundance and Ribosomal Assembly in Trypanosoma brucei

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.

The mitochondrial ATP synthase ofTrypanosoma brucei: Structure and regulation

The structure and regulation of theTrypanosoma brucei mitochondrial ATP synthase is reviewed. This enzyme complex which catalyzes the synthesis and hydrolysis of ATP within the mitochondrion is a multisubunit complex which is regulated in several ways. Several lines of evidence have shown that the ATP synthase is regulated through the life cycle ofTrypanosoma brucei. The enzyme complex is present at maximal levels in the procyclic form where mitochondrial activity is the highest and cytochromes and Krebs cycle components are present. The levels of the ATP synthase are decreased in the bloodstream forms where the levels of the mitochondrial cytochromes are absent or substantially decreased. In recent preliminary work we have shown the presence of an ATP synthase inhibitor peptide which may indicate an additional level of complexity to the regulation.

The Trypanosoma brucei mitochondrial ATP synthase is developmentally regulated at the level of transcript stability

The mitochondrial ATP synthase is developmentally regulated throughout the life cycle of the Trypanosoma brucei. The alpha and beta subunits of the F(1) moiety, and subunit 9 of the F(0) moiety of the T. brucei ATP synthase have been previously cloned and characterized. Here we have determined the chromosomal localization and developmental regulation of these three key subunits of the complex. Southern blot analysis indicates that all three of these genes are present as single copies in the T. brucei genome. Pulsed field gel analysis demonstrates that these genes are encoded in different chromosomes, and are thus not part of the same gene cluster. A comparison between the protein and steady state transcript levels for these subunits suggests that regulation of expression occurs predominantly posttranscriptionally. Comparison of mRNA stability for procyclic and bloodstream forms shows that the half life of the three transcripts is much shorter in bloodstream forms. The differences in transcript stability in the procyclic form for subunit 9 is greater than that for alpha and beta subunits, while the differences at the protein levels are comparable. These results suggest that there may be further posttranscriptional regulation of subunit 9.

Trypanosoma brucei RNA Binding Proteins p34 and p37 Mediate NOPP44/46 Cellular Localization via the Exportin 1 Nuclear Export Pathway

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.