Module 6 Assessment: ATP Synthase in Procyclic Trypanosoma brucei

This report presents an analysis of ATP synthase, an enzyme critical for energy production, focusing on its function within Procyclic Trypanosoma brucei. The study employs various experimental techniques, including plasmid construction, RNA interference (RNAi), SDS-PAGE, Western blot analysis, immunoprecipitation, immunofluorescence assays, tandem affinity purification (TAP), mass spectrometry, ATPase assays, and blue-native polyacrylamide gel electrophoresis (BN-PAGE). The research identifies homologous novel subunits and investigates the F1 subcomplex. The report details the experimental procedures, including cell culture, protein analysis, and functional assays. The findings contribute to a deeper understanding of the composition and role of ATP synthase in this organism, providing insights into its subunits and overall function in energy metabolism. The study references key publications that support the research. The report offers a comprehensive overview of the ATP synthase complex and its role in energy production in the context of the specific organism under investigation.

Module 6 Assessment

ATP Synthase
The ATP synthase refers to an enzyme which creates energy storage molecule known as
energy adenosine triphosphate and it is mostly preferred by cells as an energy currency in
different organism. The reaction catalysed by ATP Synthase is -
ADP + Pi + H+out ATP + H⇌ 2O + H+in
ATP synthase has a couple of subunits including FO and F1 which ids considered as
rotational motor mechanism for production of ATP (Lau and Rubinstein, 2012). Meanwhile,
mitochondrial FOF1 ATP synthase is considered as an essential multi- subunit protein complexed
in vast majority of eukaryotes but little is known about its composition and role of Trypanosoma
brucei.
Genes used in the study
Tb10.70.7760 (TAP_Tb7760, RNAi_Tb7760); Tb927.5.2930 (TAP_Tb2930,
RNAi_Tb2930); Tb927.5.1710 (TAP_subunit b); Tb927.3.1380 (TAP_subunit β);
Tb927.7.7430/Tb927.7.7420 (RNAi_subunit α).
SERIES OF EXPERIMENTS
Plasmid construction – To create the vectors from T. brucei strain 427 genomic DNA by
utilising several oligonucleotides which are given below:
TAP_Tb7760 Fwd – ACAAAGCTTATGCAGGGCAGTTGG
Rev – ACAGGATCCAGCTGTGTGTCGGCC
TAP_sub b Fwd – CACAAGCTTATGATGCGCCGTG
Rev – CACGGATCCCTCTACCTTTACATC
TAP_Tb2930 Fwd – ACAAAGCTTATGCGCCGTGTATC
Rev – ACAGGATCCGTGATGGGCC
TAP_sub β Fwd – ACAAAGCTTATGCTGACTCGTTTCC
Rev – ACAGGATCCGCTACTGGCTTG
In order to generate a construct of RNAi of Tb10.70.7760 and Tb927.5.2930 transcripts,
fragments of 830bp & 646bp. The clones plasmid, pZJM via Xhol and HindIII restriction sites.
RNAi_Tb7760 Fw – CACAAGCTTGAAGCTCAGGACC
Rev – CACCTCGAGGCAGAAACGCATC
RNAi_Tb2930 Fw – ACAAAGCTTATGCGCCGTGTATC
Rev – CACCTCGAGTTCGGCCCGATC
1

Cell culture and generation of cell lines – The TAP & RNAi plasmids were linearised
with Notl enzyme and transfected into cell lines which are expressed of tagged protein induced
by 100ng/ml of tet.
SDS PAGE AND Western blot analysis – The protein samples are fractionated by SDS-
PAGE, blotted onto PVDF membrane and probed with monoclonal antibodies (mAb).
Immunoprecipitation of FOF1-ATP synthase complex - In include immonuprecipitation
of FOF1- ATP synthase complex through mAb64 was performed on pooled gradient factors and
the pulled proteins can be determined via LC-MS/MS analysis (Ravera and et. al., 2011).
Immunofluorescence assay (IFA) - The subcellular localisation of tagged protein can be
identified within a cell by using IFA.
Tandem affinity purification (TAP) of tagged complexes – The TAP protocols is helpful
in purifying tagged protein and isolation of the same complexes through sequential binding to
lgG and calmodulin affinity columns.
Mass spectrometry analysis – The overall proteins were determined by multiple peptide
matches except for a couple of ATP synthase subunits including subunit ε and subunit c which
can be identified by single peptide match.
Digitonin fractionation, ATPase assay and ATP production assay – The activity of
ATPase can be measured by release of free phosphate and Crude mt preparations from RNAi
knock-down cell lines were achieved via digitotin extraction. The production of STP can be
calculated by luminometer through utilising ATP Bioluminescence assay kit CLS II.
Blue-native polyacrylamide gel electrophoresis (BN-PAGE) and histochemical staining
– The ATPase activity appears as a white precipitate with help of process by using BN-PAGE
gel which was fixed in 30% methanol (Zoschke and et. al., 2013).
The genome database of T. brucei using known subunits of mitochondrial FOF1- ATP
synthase and determine 7 homologous novel subunits which essential for Procyclic
Trypanosoma brucei. F1 sub complex contains 5 subunits designated as α, β, γ, δ, ε and they are
conserved among eukaryotes. ATP synthase subunits α (encoded by two identical open reading
frames Tb927.7.7420/Tb927.7.7430), β (Tb927.3.1380), and γ (Tb10.100.0070). Subunit δ
(Tb927.6.4990) is currently annotated as subunit ε in GeneDB.
2

REFERENCES
Books and journals
Lau, W. C. and Rubinstein, J. L., 2012. Subnanometre-resolution structure of the intact Thermus
thermophilus H+-driven ATP synthase. Nature. 481(7380). p.214.
Ravera, S. and et. al., 2011. Characterization of myelin sheath FoF1-ATP synthase and its
regulation by IF1. Cell biochemistry and biophysics. 59(2). pp.63-70.
Zoschke, R. and et. al., 2013. Mutation of the pentatricopeptide repeat-SMR protein SVR7
impairs accumulation and translation of chloroplast ATP synthase subunits in
Arabidopsis thaliana. Journal of plant research. 126(3). pp.403-414.
3