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The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle.

Duhagon MA, Pastro L, Sotelo-Silveira JR, Pérez-Díaz L, Maugeri D, Nardelli SC, Schenkman S, Williams N, Dallagiovanna B, Garat B - BMC Microbiol. (2009)

Bottom Line: However, we found that Tc38 predominantly localizes into the mitochondrion.In epimastigotes, Tc38 is found only in association with kDNA in G1 phase.In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Montevideo, Uruguay. mduhagon@fcien.edu.uy

ABSTRACT

Background: Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages.

Results: By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

Conclusions: The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

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Binding of native Tc38 to different [dT-dG] rich targets. Whole protein extracts of exponentially grown epimastigotes cultures were assayed with oligonucleotide probes representing four putative targets: TG, TEL, MIN and MAX as indicated in Materials and Methods. Reactions were done under the conditions described in Materials and Methods using 1 μg of total epimastigote protein extract, 1 ng (10,000 cpm) of each probe. For each probe, we determined the presence of Tc38 in the complexes by the addition of the anti-Tc38 affinity purified polyclonal antibody (12 μL) to the binding reactions (α-Tc38). A polyclonal antibody against TcPuf6 (12 μL) was used as a control (α-TcPuf6). The presence/absence of the antibodies and protein extract in the binding reactions is indicated by +/- signs above each lane.
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Figure 1: Binding of native Tc38 to different [dT-dG] rich targets. Whole protein extracts of exponentially grown epimastigotes cultures were assayed with oligonucleotide probes representing four putative targets: TG, TEL, MIN and MAX as indicated in Materials and Methods. Reactions were done under the conditions described in Materials and Methods using 1 μg of total epimastigote protein extract, 1 ng (10,000 cpm) of each probe. For each probe, we determined the presence of Tc38 in the complexes by the addition of the anti-Tc38 affinity purified polyclonal antibody (12 μL) to the binding reactions (α-Tc38). A polyclonal antibody against TcPuf6 (12 μL) was used as a control (α-TcPuf6). The presence/absence of the antibodies and protein extract in the binding reactions is indicated by +/- signs above each lane.

Mentions: Using EMSA we previously identified two specific complexes (TG1 and TG2) arising from the interaction of epimastigote nuclear extracts with a [dT-dG]40 oligonucleotide probe [23]. Later we also showed that the recombinant purified Tc38-GST fusion protein was able to bind the same oligonucleotide probe [12]. To directly address the participation of the endogenous Tc38 in the initially reported nuclear extract complexes we performed EMSA supershift reactions. We employed a purified polyclonal antiserum raised against the recombinant GST-Tc38 protein that specifically recognizes a main band with an apparent molecular weight of about 38 kDa in total protein extracts of epimastigotes (see below). This antibody was able to supershift the complexes formed by the recombinant GST-Tc38 protein and the poly [dT-dG] probe (data not shown). As seen in Figure 1, complexes TG1 and TG2 were readily supershifted by this antibody. No supershift could be observed using the complementary oligonucleotide [dC-dA]40 as a probe (data not shown). These data indicate that Tc38 is present in the native protein complexes formed between the poly [dT-dG] probe and parasite extracts characterized previously [23] and favors its role in the in vivo sequence recognition.


The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle.

Duhagon MA, Pastro L, Sotelo-Silveira JR, Pérez-Díaz L, Maugeri D, Nardelli SC, Schenkman S, Williams N, Dallagiovanna B, Garat B - BMC Microbiol. (2009)

Binding of native Tc38 to different [dT-dG] rich targets. Whole protein extracts of exponentially grown epimastigotes cultures were assayed with oligonucleotide probes representing four putative targets: TG, TEL, MIN and MAX as indicated in Materials and Methods. Reactions were done under the conditions described in Materials and Methods using 1 μg of total epimastigote protein extract, 1 ng (10,000 cpm) of each probe. For each probe, we determined the presence of Tc38 in the complexes by the addition of the anti-Tc38 affinity purified polyclonal antibody (12 μL) to the binding reactions (α-Tc38). A polyclonal antibody against TcPuf6 (12 μL) was used as a control (α-TcPuf6). The presence/absence of the antibodies and protein extract in the binding reactions is indicated by +/- signs above each lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654453&req=5

Figure 1: Binding of native Tc38 to different [dT-dG] rich targets. Whole protein extracts of exponentially grown epimastigotes cultures were assayed with oligonucleotide probes representing four putative targets: TG, TEL, MIN and MAX as indicated in Materials and Methods. Reactions were done under the conditions described in Materials and Methods using 1 μg of total epimastigote protein extract, 1 ng (10,000 cpm) of each probe. For each probe, we determined the presence of Tc38 in the complexes by the addition of the anti-Tc38 affinity purified polyclonal antibody (12 μL) to the binding reactions (α-Tc38). A polyclonal antibody against TcPuf6 (12 μL) was used as a control (α-TcPuf6). The presence/absence of the antibodies and protein extract in the binding reactions is indicated by +/- signs above each lane.
Mentions: Using EMSA we previously identified two specific complexes (TG1 and TG2) arising from the interaction of epimastigote nuclear extracts with a [dT-dG]40 oligonucleotide probe [23]. Later we also showed that the recombinant purified Tc38-GST fusion protein was able to bind the same oligonucleotide probe [12]. To directly address the participation of the endogenous Tc38 in the initially reported nuclear extract complexes we performed EMSA supershift reactions. We employed a purified polyclonal antiserum raised against the recombinant GST-Tc38 protein that specifically recognizes a main band with an apparent molecular weight of about 38 kDa in total protein extracts of epimastigotes (see below). This antibody was able to supershift the complexes formed by the recombinant GST-Tc38 protein and the poly [dT-dG] probe (data not shown). As seen in Figure 1, complexes TG1 and TG2 were readily supershifted by this antibody. No supershift could be observed using the complementary oligonucleotide [dC-dA]40 as a probe (data not shown). These data indicate that Tc38 is present in the native protein complexes formed between the poly [dT-dG] probe and parasite extracts characterized previously [23] and favors its role in the in vivo sequence recognition.

Bottom Line: However, we found that Tc38 predominantly localizes into the mitochondrion.In epimastigotes, Tc38 is found only in association with kDNA in G1 phase.In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Montevideo, Uruguay. mduhagon@fcien.edu.uy

ABSTRACT

Background: Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages.

Results: By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

Conclusions: The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

Show MeSH
Related in: MedlinePlus