Ivor Tittawella

Protein synthesis in mitochondria isolated from the trypanosomatid protozoan Crithidia fasciculata

Evidence presented over the years in support of mitochondrial translation in trypanosomes, based largely on studies using differential inhibitors such as cycloheximide and chloramphenicol, remains controversial. I have studied endogenous mRNA‐dependent translation in a mitochondrial fraction isolated from the trypanosomatid protozoan Crithidia fasciculata. By using pancreatic ribonuclease to inactivate contaminating cytosolic activity, I show that these mitochondria can conduct protein synthesis in their own right. The mitochondrial translational products differed from cytosolic products as judged by SDS‐PAGE, and had sizes expected of some proteins encoded in the mitochondrial genome of C. fasciculata and other trypanosomatids. Some evidence is provided suggesting that the seat of translation might be the kinetoplast.

Two proteins involved in kinetoplast compaction

The kinetoplast is the genome of the single mitochondrion of trypanosomatid Protozoa, and contains up to 30% of total cellular DNA in a network of catenated AT‐rich rings. EM studies show that the kinetoplast is organized into a compact, disc‐shaped structure in vivo, but little is known about proteins involved in its architecture. Defining such proteins would be useful to understand the molecular biology of this unusual organelle and to design compounds to contain parasite growth. We show here that two proteins, p1 and p2 of M r ~ 22 and ~ 21 kDa, respectively, from the trypanosomatid Crithidia fasciculata can compact kDNA networks efficiently in vitro, the first such demonstration with purified trypanosome proteins. We show that these proteins are localized exclusively in the parasites kinetoplast. Our data thus define two proteins potentially involved in kinetoplast organization in vivo.

Rifampicin-induced protein synthesis: A pre-requisite for increased expression of the ββ′ operon in Escherichia coli

SummaryIn a rifS/rifR heterodiploid strain of E. coli, a 4 minute pulse of rifampicin can induce a prolonged (>60 min) increase in the rate of synthesis of the RNA polymerase subunits, β and β′. The application of a constraint on the fidelity of protein synthesis during, but not after, the rifampicin pulse partially arrests the development of this capacity for subunit synthesis. I discuss the implications of these findings in relation to the control of the ββ′ operon in E. coli.

Mitochondrial ribosomes in a trypanosome.

The nature, and even the existence, of trypanosome mitochondrial ribosomes has been the subject of some debate. We investigated this further in the insect trypanosome, Crithidia fasciculata. In sucrose gradients of parasite lysates, mitochondrial ribosomal RNA co-sediments at approximately 35S with nascent peptides synthesized in the presence of the cytosolic translational inhibitor, cycloheximide. Co-sedimenting peptides in this peak are much reduced when the parasites are treated with the bacterial translational inhibitor, chloramphenicol. In CsCl gradients this peak resolves at a buoyant density of 1.42 g/cm(3), a value typical for mito-ribosomes. Electron microscopy of peak material shows particles smaller than cytosolic ribosomes, but with characteristic ribosomal shapes. We propose that these particles represent the parasites mitochondrial ribosomes.

Kinetoplast DNA-aggregating proteins from the parasitic protozoan Crithidia fasciculata.

Proteins from C. fasciculata, which preferentially aggregate the parasites AT‐rich mitochondrial (kinetoplast) DNA in vitro, are reported. The aggregation is non‐topological, non‐covalent, independent of Mg2+ and ATP, but much improved by spennidine. We discuss how these proteins might modulate the shape and size of the parasites mitochondrial genome.

Localization of riboproteins in a trypanosomatid mitochondrion

There is growing evidence in support of mitochondrial translation in trypanosomes but mitoribosomes have never been characterized or localized in these parasites. On RNA–protein blots we identified several proteins from the trypanosomatid Crithidia fasciculata which bound the parasites 12S and 9S mitochondrial ribosomal RNAs. Two of these proteins had significant amino acid sequence homology to riboproteins S8 and S21 across phyla. Immunoelectron microscopy revealed that antibodies raised against the two proteins react with matrix components in the C. fasciculata mitochondrion. Our data thus provide, we believe for the first time, evidence for the presence of riboproteins within a trypanosomatid mitochondrion, bound, possibly, to the 12S and 9S RNAs. The proteins were immunologically related to two cytosolic riboproteins which were also of identical size, suggesting the interesting possibility that the same set of riboproteins is shared between the cytosol and the mitochondrion in this parasite.

Variable RNA polymerase populations in the life cycle of Trypanosoma brucei

DNA‐dependent RNA polymerase from blood forms and culture forms of the parasitic protozoan Trypanosoma brucei was resolved into multiple peaks of activity by DEAE‐Sephadex chromatography. The enzyme from the two forms was found to be different by several criteria, suggesting that it is subject to developmental control.

Mutant of Escherichia coli with unusual patterns of rpoB,C expression in response to rifampicin and acridine orange.

SummaryA mutation located near rpoB (89′) in E. coli is responsible for unusual patterns of β and β′ (but not L7/L12) synthesis in response to the drugs rifampicin and acridine orange.