Identification and characterization of three peroxins--PEX6, PEX10 and PEX12--involved in glycosome biogenesis in Trypanosoma brucei

Protozoan Kinetoplastida such as the pathogenic trypanosomes compartmentalize several important metabolic systems, including the glycolytic pathway, in peroxisome-like organelles designated glycosomes. Genes for three proteins involved in glycosome biogenesis of Trypanosoma brucei were identified. A preliminary analysis of these proteins, the peroxins PEX6, PEX10 and PEX12, was performed. Cellular depletion of these peroxins by RNA interference affected growth of both mammalian bloodstream-form and insect-form (procyclic) trypanosomes. The bloodstream forms, which rely entirely on glycolysis for their ATP supply, were more rapidly killed. Both by immunofluorescence studies of intact procyclic T. brucei cells and subcellular fractionation experiments involving differential permeabilization of plasma and organellar membranes it was shown that RNAi-dependent knockdown of the expression of each of these peroxins resulted in the partial mis-localization of different types of glycosomal matrix enzymes to the cytoplasm: proteins with consensus motifs such as the C-terminal type 1 peroxisomal targeting signal PTS1 or the N-terminal signal PTS2 and a protein for which the sorting information is present in a polypeptide-internal fragment not containing an identifiable consensus sequence.     

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.     

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.     

Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.     

Biogenesis of the glycosome in Trypanosoma brucei: the synthesis, translocation and turnover of glycosomal polypeptides.

Glycosomes, the microbodies of Trypanosoma brucei, contain a number of enzymes involved in glucose and glycerol metabolism. The biogenesis of three of these enzymes has been studied. Aldolase, D-glyceraldehyde-3-phosphate dehydrogenase and NAD-linked glycerol-3-phosphate dehydrogenase are all synthesized in the cytosol on free rather than on membrane-bound polysomes. In vitro, as well as in vivo, these polypeptides are synthesized at their mature size, and no evidence was found for any processing upon entry into the glycosomes. Continuous and pulse-chase labelling experiments with procyclic trypomastigotes revealed that the enzymes have a half-life in the cytosol of approximately 3 min or less, and then turn over rapidly in the glycosomes, with half-lives as short as 30 min.     

Intermolecular interactions involved in the association of the variant surface glycoprotein of Trypanosoma brucei

Trypanosomes in their mammalian host are covered by the densely packed variant surface glycoprotein (VSG). Depending on the presence or absence of a glycosyl-phosphatidyl inositol anchor. VSG is accessible as soluble globular protein (sVSG), or as insoluble membrane form (mfVSG). In order to get insight into the two-dimensional association of VSG within the surface layer, protein-protein interactions were investigated in a wide range of protein concentrations. No self-assembly of sVSG could be detected even at protein concentrations close to the local packing in the surface layer. The absence of preferential interactions with soybean phospholipid or lysolecithin monolayers (spread on a Langmuir trough) suggests that the soluble form of the protein is not integrated into a model lipid-water interface. Thus, the two-dimensional arrangement of the protein in situ seems to be determined by hydrophobic interactions of the lipid components rather than protein-lipid interactions. In contrast to sVSG, the membrane form (mfVSG) undergoes aggregation and shows a strong tendency to absorb to surfaces and chromatographic matrices, thus interfering with standard techniques of protein purification.     

Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.     

The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.     

The KREPA3 zinc finger motifs and OB-fold domain are essential for RNA editing and survival of Trypanosoma brucei

Three types of editosomes, each with an identical core containing six related KREPA proteins, catalyze the U insertion and deletion RNA editing of mitochondrial mRNAs in trypanosomes. Repression of expression of one of these, KREPA3 (also known as TbMP42), shows that it is essential for growth and in vivo editing in both procyclic (PF) and bloodstream (BF) life cycle stages of Trypanosoma brucei. RNA interference knockdown results in editosome disruption and altered in vitro editing in PFs, while repression by regulatable double knockout results in almost complete loss of editosomes in BFs. Mutational analysis shows that the KREPA3 zinc fingers and OB-fold domain are each essential for growth and in vivo editing. Nevertheless, KREPA3 with mutated zinc fingers incorporates into editosomes that catalyze in vitro editing and thus is not essential for editosome integrity, although stability is affected. In contrast, the OB-fold domain is essential for editosome integrity. Overall, KREPA3, especially its OB-fold, functions in editosome integrity, and its zinc fingers are essential for editing in vivo but not for the central catalytic steps. KREPA3 may function in editosome organization and/or RNA positioning.     

Protein-protein interactions in the membrane: sequence, structural, and biological motifs

Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.     

Two trypanosome-specific proteins are essential factors for 5S rRNA abundance and ribosomal assembly in Trypanosoma brucei

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 PARP and rRNA promoters of Trypanosoma brucei are composed of dissimilar sequence elements that are functionally interchangeable.

The African trypanosomes express two major surface proteins, the variant surface glycoprotein (VSG) and the procyclic acidic repetitive protein (PARP). The RNA polymerase that transcribes the VSG and PARP genes shares many characteristics with RNA polymerase I. We show that although there is very little similarity in nucleotide sequence, the functional structure of a trypanosome rRNA promoter is almost identical to that of the PARP promoter. Further, domains from the PARP promoter can functionally substitute for the corresponding parts of the rRNA promoter, and vice versa.     

An evolutionarily conserved coiled-coil protein implicated in polycystic kidney disease is involved in basal body duplication and flagellar biogenesis in Trypanosoma brucei

Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.     

Partial amino acid sequence and functional aspects of histone H1 proteins in Trypanosoma brucei brucei

Summary— Trypanosoma brucei brucei, a protozoan parasite of wild and domestic animals in Africa, is related to the pathogenic agent of human sleeping sickness. Four H1 histone proteins were isolated from nuclei of procyclic culture forms and cleaved with proteases. Amino acid sequence analysis of purified fragments indicated the presence of variants which displayed sequence identities as compared to the C-terminal domain of human H1. Substitutions of amino acids and posttranslational modifications of the histones in iT b brucei H1 may influence protein conformation and histone-histone as well as histone-DNA interactions in the chromatin of the parasite. Digestion of soluble chromatin with immobilized trypsin at low and high ionic strengths indicated an internal localization of H1 in the condensed chromatin. The influence of histone H1 of T b brucei on the compaction pattern of the chromatin was investigated by dissociation and reconstitution experiments. Electron microscopy revealed that trypanosome H1 was able to induce condensation of the chromatin of the parasite and of rat liver into dense tangles. After dephosphorylation of H1, 30 nm fibers were induced in rat liver chromatin, while the resulting fibers were distinctly thinner in T b brucei. It can be concluded that the absence of 30 nm fibers in T b brucei chromatin cannot be explained by the divergent variants and posttranslational phosphorylations of H1 only but rather by the influence of both, the divergent core histones, previously described, and H1 properties.     

Multiple motifs regulate trafficking of the LAMP-like protein p67 in the ancient eukaryote Trypanosoma brucei

p67 is a lysosome-associated membrane protein-like lysosomal type I transmembrane glycoprotein in African trypanosomes. The p67 cytoplasmic domain (CD) is both necessary and sufficient for lysosomal targeting in procyclic insect-stage parasites. The p67CD contains two [DE]XXXL[LI]-type dileucine motifs, which function as lysosomal targeting signals in mammalian cells. Using a green fluorescent protein fusion to the p67 transmembrane and cytoplasmic domains as a reporter system, we investigated the role of these motifs in lysosomal targeting in procyclic trypanosomes. Pulse-chase turnover studies, steady-state immunolocalization and quantitative flow cytometry all gave consistent results. Mutagenesis of the membrane-distal dileucine motif impairs lysosomal trafficking leading to partial appearance of the reporter on the cell surface. Mutagenesis of the membrane-proximal motif has little effect on proper targeting. Simultaneous mutagenesis of both motifs results in quantitative delivery to the cell surface. Thus, the distal motif plays a dominant role, but both dileucine motifs are necessary for maximal lysosomal targeting. Additional studies suggest that the upstream acidic residues in each motif influence lysosomal targeting and may also affect forward trafficking in the early secretory pathway. These results strongly suggest an evolutionary conservation in lysosomal trafficking mechanisms in the ancient eukaryote Trypanosoma brucei.     

A glycosomal protein (p60) which is predominantly expressed in procyclic Trypanosoma brucei. Characterization and DNA sequence

Glycosomes are specialized organelles of trypanosomes which contain glycolytic enzymes as their major protein components in Trypanosoma brucei bloodstream form. In the glycosomes of the insect form of T. brucei, additional enzyme activities are found but have not yet been ascribed to a particular protein molecule. In this study, we report the characterization of a 60-kDa glycosomal protein (p60) encoded by a single copy gene which is transcribed into a mRNA of 2.9 kilobases. The gene codes for a protein of 472 amino acids with a molecular mass of 52.5 kDa, suggesting that the mRNA contains large untranslated regions of about 1.4 kilobases. Genomic DNA hybridizations have shown that the gene for p60 is confined to the family of Trypanosomatidae. Sequence comparison confirmed that p60 is not a member of a conserved protein family and does not belong to the group of glycolytic enzymes. p60 is expressed much more strongly in insect form than in bloodstream form trypanosomes. Thus, p60 is the first glycosomal protein observed whose expression is up-regulated during the transition of trypanosomes from the bloodstream to the insect form. The biochemical characterization of p60 demonstrated its capability to bind microtubules and membrane vesicles and to cross-link these structures. These properties might indicate a function in linking glycosomes to the microtubules of the trypanosomal cytoskeleton. However, proteinase K digestion experiments indicate that p60 is not exposed at the outer surface of the glycosomal membrane. The biological role of the microtubule-binding capability of p60 remains unclear, whereas its membrane binding may be of physiological significance inside the glycosome.     

KREPB6, KREPB7, and KREPB8 are important for editing endonuclease function in Trypanosoma brucei

Three distinct editosomes are required for the uridine insertion/deletion editing that creates translatable mitochondrial mRNAs in Trypanosoma brucei. They contain KREPB6, KREPB7, or KREPB8 proteins and their respective endonucleases KREN3, KREN2, or KREN1. RNAi knockdowns of KREPB6, KREPB7, and KREPB8 variably affect growth and RNA editing. KREPB6 and KREPB7 knockdowns substantially reduced in vitro insertion site cleavage activity of their respective editosomes, while KREPB8 knockdown did not affect its editosome deletion site cleavage activity despite inhibition of growth and editing. KREPB6, KREPB7, and KREPB8 knockdowns disrupted tagged KREN3, KREN2, or KREN1 editosomes, respectively, to varying degrees, and in the case of KREN1 editosomes, the deletion editing site cleavage activity shifted to a smaller S value. The varying effects correlate with a combination of the relative abundances of the KREPB6-8 proteins and of the different insertion and deletion sites. Tagged KREPB6-8 were physically associated with deletion subcomplexes upon knockdown of the centrally interactive KREPA3 protein, while KREN1-3 endonucleases were associated with insertion subcomplexes. The results indicate that KREPB6-8 occupy similar positions in editosomes and are important for the activity and specificity of their respective endonucleases. This suggests that they contribute to the accurate recognition of the numerous similar but diverse editing site substrates.