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Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus.

Russo I, Oksman A, Goldberg DE - Mol. Microbiol. (2009)

Bottom Line: Palmitoylation status has an important role in dictating P. falciparum calpain localization.The targeting signals function in mammalian cells as well as in the parasite.P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Washington University School of Medicine, Department of Molecular Microbiology, St Louis, Missouri 63110, USA.

ABSTRACT
The Plasmodium falciparum genome encodes a single calpain. By generating P. falciparum clones expressing C-terminally tagged calpain, we localized this protein to the nucleolus. Pf_calpain possesses an unusual and long N-terminal domain in which we identified three subregions that are highly conserved among Plasmodium species. Two have putative targeting signals: a myristoylation motif and a nuclear localization sequence. We assessed their functionality. Our data show that the nuclear localization sequence is an active nuclear import motif that contains an embedded signal conferring nucleolar localization on various chimeras. The N-terminus is myristoylated at Gly2 and palmitoylated at Cys3 and Cys22. Palmitoylation status has an important role in dictating P. falciparum calpain localization. The targeting signals function in mammalian cells as well as in the parasite. P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.

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Related in: MedlinePlus

Immunofluorescence and EM analyses reveal that Pf_calpain is nucleolar. A. Colocalization of calpain and Nop1, a nucleolar marker. Using specific antibodies for GFP or for flag epitope in combination with proper AF488-conjugated secondary antibodies, we detected the cellular distribution of calpain in the clones expressing calpain-GFP and calpain-2×-flag in relation to the nucleolar compartment labelling obtained with an anti-Pf_Nop1 serum (red channel). The nuclei are stained with DAPI. Bar, 5 μm. Enlargements of the nuclei are provided in Fig. S3. B. Immuno-EM was done using pre-embedding labelling with anti-flag and anti-Pf_Nop1 after tetanolysin treatment to permeabilize erythrocytes. The image presents the nuclear section of a representative early trophozoite. At the upper left corner are visible the red blood cell (R) and a cytostome (C). The nucleus (N) appears thoroughly circumscribed by its double membrane closely associated with ER (E). Inside the nucleus, labelling due to the immunodetection of calpain-flag (gold particles 12 nm – open arrows in the enlarged panel) and Pf_Nop1 (gold particles 18 nm – solid arrows in the enlarged panel) reveals staining of an electron-dense nuclear subcompartment. Some particles are also found in the perinuclear ER (asterisk). Bars, 0.5 μm. C. Confocal analysis of purified nuclei from 3D7 using anti-calpain antiserum #35. We generally detected one or two spots of approximate diameter of 0.5 μm. Colocalizazion of Nop1 and calpain in an isolated nucleus by confocal analysis is provided in Fig. S3.
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fig03: Immunofluorescence and EM analyses reveal that Pf_calpain is nucleolar. A. Colocalization of calpain and Nop1, a nucleolar marker. Using specific antibodies for GFP or for flag epitope in combination with proper AF488-conjugated secondary antibodies, we detected the cellular distribution of calpain in the clones expressing calpain-GFP and calpain-2×-flag in relation to the nucleolar compartment labelling obtained with an anti-Pf_Nop1 serum (red channel). The nuclei are stained with DAPI. Bar, 5 μm. Enlargements of the nuclei are provided in Fig. S3. B. Immuno-EM was done using pre-embedding labelling with anti-flag and anti-Pf_Nop1 after tetanolysin treatment to permeabilize erythrocytes. The image presents the nuclear section of a representative early trophozoite. At the upper left corner are visible the red blood cell (R) and a cytostome (C). The nucleus (N) appears thoroughly circumscribed by its double membrane closely associated with ER (E). Inside the nucleus, labelling due to the immunodetection of calpain-flag (gold particles 12 nm – open arrows in the enlarged panel) and Pf_Nop1 (gold particles 18 nm – solid arrows in the enlarged panel) reveals staining of an electron-dense nuclear subcompartment. Some particles are also found in the perinuclear ER (asterisk). Bars, 0.5 μm. C. Confocal analysis of purified nuclei from 3D7 using anti-calpain antiserum #35. We generally detected one or two spots of approximate diameter of 0.5 μm. Colocalizazion of Nop1 and calpain in an isolated nucleus by confocal analysis is provided in Fig. S3.

Mentions: The best-characterized subnuclear compartment is the nucleolus, known as the site of rRNA transcription, ribosome and ribonucleoprotein complex assembly and protein sequestration (Birnstiel et al., 1963; Maggi and Weber, 2005). We performed dual staining using an antiserum to the well-studied nucleolar marker Pf_Nop1 (Figueiredo et al., 2005), in combination with anti-flag or anti-GFP antibodies. The results (Fig. 3A and Fig. S3) show a clear colocalization of Pf_Nop1 with calpain-GFP (a), as well as with calpain-2×-flag (b). Similar colocalization was also obtained using anti-human Nop1 (60% identity to Pf_Nop1, data not shown). Signal with anti-tag antibodies could be detected in late rings and early to mid-trophozoites; a nucleolar focus was seen throughout this developmental period.


Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus.

Russo I, Oksman A, Goldberg DE - Mol. Microbiol. (2009)

Immunofluorescence and EM analyses reveal that Pf_calpain is nucleolar. A. Colocalization of calpain and Nop1, a nucleolar marker. Using specific antibodies for GFP or for flag epitope in combination with proper AF488-conjugated secondary antibodies, we detected the cellular distribution of calpain in the clones expressing calpain-GFP and calpain-2×-flag in relation to the nucleolar compartment labelling obtained with an anti-Pf_Nop1 serum (red channel). The nuclei are stained with DAPI. Bar, 5 μm. Enlargements of the nuclei are provided in Fig. S3. B. Immuno-EM was done using pre-embedding labelling with anti-flag and anti-Pf_Nop1 after tetanolysin treatment to permeabilize erythrocytes. The image presents the nuclear section of a representative early trophozoite. At the upper left corner are visible the red blood cell (R) and a cytostome (C). The nucleus (N) appears thoroughly circumscribed by its double membrane closely associated with ER (E). Inside the nucleus, labelling due to the immunodetection of calpain-flag (gold particles 12 nm – open arrows in the enlarged panel) and Pf_Nop1 (gold particles 18 nm – solid arrows in the enlarged panel) reveals staining of an electron-dense nuclear subcompartment. Some particles are also found in the perinuclear ER (asterisk). Bars, 0.5 μm. C. Confocal analysis of purified nuclei from 3D7 using anti-calpain antiserum #35. We generally detected one or two spots of approximate diameter of 0.5 μm. Colocalizazion of Nop1 and calpain in an isolated nucleus by confocal analysis is provided in Fig. S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2746569&req=5

fig03: Immunofluorescence and EM analyses reveal that Pf_calpain is nucleolar. A. Colocalization of calpain and Nop1, a nucleolar marker. Using specific antibodies for GFP or for flag epitope in combination with proper AF488-conjugated secondary antibodies, we detected the cellular distribution of calpain in the clones expressing calpain-GFP and calpain-2×-flag in relation to the nucleolar compartment labelling obtained with an anti-Pf_Nop1 serum (red channel). The nuclei are stained with DAPI. Bar, 5 μm. Enlargements of the nuclei are provided in Fig. S3. B. Immuno-EM was done using pre-embedding labelling with anti-flag and anti-Pf_Nop1 after tetanolysin treatment to permeabilize erythrocytes. The image presents the nuclear section of a representative early trophozoite. At the upper left corner are visible the red blood cell (R) and a cytostome (C). The nucleus (N) appears thoroughly circumscribed by its double membrane closely associated with ER (E). Inside the nucleus, labelling due to the immunodetection of calpain-flag (gold particles 12 nm – open arrows in the enlarged panel) and Pf_Nop1 (gold particles 18 nm – solid arrows in the enlarged panel) reveals staining of an electron-dense nuclear subcompartment. Some particles are also found in the perinuclear ER (asterisk). Bars, 0.5 μm. C. Confocal analysis of purified nuclei from 3D7 using anti-calpain antiserum #35. We generally detected one or two spots of approximate diameter of 0.5 μm. Colocalizazion of Nop1 and calpain in an isolated nucleus by confocal analysis is provided in Fig. S3.
Mentions: The best-characterized subnuclear compartment is the nucleolus, known as the site of rRNA transcription, ribosome and ribonucleoprotein complex assembly and protein sequestration (Birnstiel et al., 1963; Maggi and Weber, 2005). We performed dual staining using an antiserum to the well-studied nucleolar marker Pf_Nop1 (Figueiredo et al., 2005), in combination with anti-flag or anti-GFP antibodies. The results (Fig. 3A and Fig. S3) show a clear colocalization of Pf_Nop1 with calpain-GFP (a), as well as with calpain-2×-flag (b). Similar colocalization was also obtained using anti-human Nop1 (60% identity to Pf_Nop1, data not shown). Signal with anti-tag antibodies could be detected in late rings and early to mid-trophozoites; a nucleolar focus was seen throughout this developmental period.

Bottom Line: Palmitoylation status has an important role in dictating P. falciparum calpain localization.The targeting signals function in mammalian cells as well as in the parasite.P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Washington University School of Medicine, Department of Molecular Microbiology, St Louis, Missouri 63110, USA.

ABSTRACT
The Plasmodium falciparum genome encodes a single calpain. By generating P. falciparum clones expressing C-terminally tagged calpain, we localized this protein to the nucleolus. Pf_calpain possesses an unusual and long N-terminal domain in which we identified three subregions that are highly conserved among Plasmodium species. Two have putative targeting signals: a myristoylation motif and a nuclear localization sequence. We assessed their functionality. Our data show that the nuclear localization sequence is an active nuclear import motif that contains an embedded signal conferring nucleolar localization on various chimeras. The N-terminus is myristoylated at Gly2 and palmitoylated at Cys3 and Cys22. Palmitoylation status has an important role in dictating P. falciparum calpain localization. The targeting signals function in mammalian cells as well as in the parasite. P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.

Show MeSH
Related in: MedlinePlus