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Crystal structure of malaria parasite nucleosome assembly protein: distinct modes of protein localization and histone recognition.

Gill J, Yogavel M, Kumar A, Belrhali H, Jain SK, Rug M, Brown M, Maier AG, Sharma A - J. Biol. Chem. (2009)

Bottom Line: Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm.A detailed analysis of PfNapL structure suggests unique histone binding properties.The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India.

ABSTRACT
Nucleosome assembly proteins (NAPs) are histone chaperones that are essential for the transfer and incorporation of histones into nucleosomes. NAPs participate in assembly and disassembly of nucleosomes and in chromatin structure organization. Human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain structural insights into the mechanism of NAPs, we have determined and analyzed the crystal structure of PfNapL at 2.3 A resolution. PfNapL, an ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b (beta)/INHAT. The PfNapL monomer is comprised of domain I, containing a dimerization alpha-helix, and a domain II, composed of alpha-helices and a beta-subdomain. Structural comparisons reveal that the "accessory domain," which is inserted between the domain I and domain II in yeast NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene were not successful, indicating its essential role for the malaria parasite. A detailed analysis of PfNapL structure suggests unique histone binding properties. The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

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Stage specific expression of PfNapL-GFP chimeras. a, Western blot of transgenic cell lines expressing GFP-tagged versions of NapL with antibodies against GFP. Expression of PfHsp70 was detected to ensure similar loading. The localization of PfNapL wild type-GFP (b), PfNapL L46A-GFP (c), and PfNapL Q50A (d) in different stages of the erythrocytic life cycle of P. falciparum is shown. The first column of each panel shows bright field pictures, followed by a nuclear DAPI stain, GFP fluorescence, an overlay of the nuclear and GFP localization, and (in the last column) overlay of bright field with DAPI and GFP.
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fig4: Stage specific expression of PfNapL-GFP chimeras. a, Western blot of transgenic cell lines expressing GFP-tagged versions of NapL with antibodies against GFP. Expression of PfHsp70 was detected to ensure similar loading. The localization of PfNapL wild type-GFP (b), PfNapL L46A-GFP (c), and PfNapL Q50A (d) in different stages of the erythrocytic life cycle of P. falciparum is shown. The first column of each panel shows bright field pictures, followed by a nuclear DAPI stain, GFP fluorescence, an overlay of the nuclear and GFP localization, and (in the last column) overlay of bright field with DAPI and GFP.

Mentions: In order to address whether PfNapL is located at some stage in the nucleus and whether its cytosolic localization is due to continuous export mediated by its putative NES, we generated cell lines expressing PfNapL-GFP chimeras. For these chimeras, we either used wild-type sequence or introduced mutations in the conserved residues of the NES (PfNapL L46A or PfNapL Q50A) (Fig. 3c). Disruption of a functional NES was predicted to lead to the accumulation of GFP chimera in the parasite nucleus. Expression of the GFP chimeras was verified via Western blot and probed with anti-GFP antibodies (Fig. 4a). A band of ∼70 kDa was observed in all cell lines, corresponding to the size of PfNapL (40.5 kDa) fused to GFP (26.8 kDa). The GFP fusion protein of all three transfectants (wild type and mutants) was detectable in the cytoplasm of all stages. Previous localization experiments with PfNapL-specific antibodies detected an exclusive cytoplasmic localization in all stages, except for the schizont stages, where the separation between nuclear (DAPI) and PfNapL staining was less defined (13). This could be due to greater interdigitation of the nucleus and cytoplasm at that stage or due to a closer association of the PfNapL pool with the nucleus. To address this question, we followed the localization of PfNapL wild type over the asexual erythrocytic life cycle (Fig. 4, b-d). PfNapL wild type-GFP was detected in the cytoplasm of the parasites. No fluorescence could be detected in the erythrocyte cytosol. Neither the food vacuole nor the nucleus of the parasite showed fluorescence in any parasite stage. This indicates that PfNapL is exclusively located in the cytoplasm of the parasite and that its putative NES is not necessary for its localization, and the predicted NES is not functional/relevant in PfNapL.


Crystal structure of malaria parasite nucleosome assembly protein: distinct modes of protein localization and histone recognition.

Gill J, Yogavel M, Kumar A, Belrhali H, Jain SK, Rug M, Brown M, Maier AG, Sharma A - J. Biol. Chem. (2009)

Stage specific expression of PfNapL-GFP chimeras. a, Western blot of transgenic cell lines expressing GFP-tagged versions of NapL with antibodies against GFP. Expression of PfHsp70 was detected to ensure similar loading. The localization of PfNapL wild type-GFP (b), PfNapL L46A-GFP (c), and PfNapL Q50A (d) in different stages of the erythrocytic life cycle of P. falciparum is shown. The first column of each panel shows bright field pictures, followed by a nuclear DAPI stain, GFP fluorescence, an overlay of the nuclear and GFP localization, and (in the last column) overlay of bright field with DAPI and GFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665062&req=5

fig4: Stage specific expression of PfNapL-GFP chimeras. a, Western blot of transgenic cell lines expressing GFP-tagged versions of NapL with antibodies against GFP. Expression of PfHsp70 was detected to ensure similar loading. The localization of PfNapL wild type-GFP (b), PfNapL L46A-GFP (c), and PfNapL Q50A (d) in different stages of the erythrocytic life cycle of P. falciparum is shown. The first column of each panel shows bright field pictures, followed by a nuclear DAPI stain, GFP fluorescence, an overlay of the nuclear and GFP localization, and (in the last column) overlay of bright field with DAPI and GFP.
Mentions: In order to address whether PfNapL is located at some stage in the nucleus and whether its cytosolic localization is due to continuous export mediated by its putative NES, we generated cell lines expressing PfNapL-GFP chimeras. For these chimeras, we either used wild-type sequence or introduced mutations in the conserved residues of the NES (PfNapL L46A or PfNapL Q50A) (Fig. 3c). Disruption of a functional NES was predicted to lead to the accumulation of GFP chimera in the parasite nucleus. Expression of the GFP chimeras was verified via Western blot and probed with anti-GFP antibodies (Fig. 4a). A band of ∼70 kDa was observed in all cell lines, corresponding to the size of PfNapL (40.5 kDa) fused to GFP (26.8 kDa). The GFP fusion protein of all three transfectants (wild type and mutants) was detectable in the cytoplasm of all stages. Previous localization experiments with PfNapL-specific antibodies detected an exclusive cytoplasmic localization in all stages, except for the schizont stages, where the separation between nuclear (DAPI) and PfNapL staining was less defined (13). This could be due to greater interdigitation of the nucleus and cytoplasm at that stage or due to a closer association of the PfNapL pool with the nucleus. To address this question, we followed the localization of PfNapL wild type over the asexual erythrocytic life cycle (Fig. 4, b-d). PfNapL wild type-GFP was detected in the cytoplasm of the parasites. No fluorescence could be detected in the erythrocyte cytosol. Neither the food vacuole nor the nucleus of the parasite showed fluorescence in any parasite stage. This indicates that PfNapL is exclusively located in the cytoplasm of the parasite and that its putative NES is not necessary for its localization, and the predicted NES is not functional/relevant in PfNapL.

Bottom Line: Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm.A detailed analysis of PfNapL structure suggests unique histone binding properties.The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India.

ABSTRACT
Nucleosome assembly proteins (NAPs) are histone chaperones that are essential for the transfer and incorporation of histones into nucleosomes. NAPs participate in assembly and disassembly of nucleosomes and in chromatin structure organization. Human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain structural insights into the mechanism of NAPs, we have determined and analyzed the crystal structure of PfNapL at 2.3 A resolution. PfNapL, an ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b (beta)/INHAT. The PfNapL monomer is comprised of domain I, containing a dimerization alpha-helix, and a domain II, composed of alpha-helices and a beta-subdomain. Structural comparisons reveal that the "accessory domain," which is inserted between the domain I and domain II in yeast NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene were not successful, indicating its essential role for the malaria parasite. A detailed analysis of PfNapL structure suggests unique histone binding properties. The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

Show MeSH
Related in: MedlinePlus