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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 localization of calpain. A. Immunofluorescence images of clones expressing calpain-6-his, calpain-GFP and calpain-2×-flag are presented. The images are representative of the overall population and were obtained using tag-specific primary antibodies and a proper secondary antibody conjugated to Alexa-Fluor (AF) 488. The immunofluorescence distribution in all the clones highlights a region closely associated with the nucleus but not completely merging with the DAPI signal. B. Colocalization of fluorescence signal distributions from both anti-flag antibody and anti-calpain antibody with that of a C-terminal peptide of calpain (#35, Fig. S1). Images a and b show the calpain-2×-flag clone stained with monoclonal anti-flag antibody/goat AF488-conjugated anti-mouse antibody and either rabbit anti-calpain #35/goat AF555-conjugated anti-rabbit or pre-immune serum of the same rabbit/goat AF555-conjugated anti-rabbit. The 3D7 parental strain, stained as in a, is positive with anti-calpain #35, but not with the anti-flag antibody. C. The relative distributions of calpain-2×-flag (green channel) and BiP (red channel) signals were analysed by confocal microscopy. Here two representative images show the signal detected in a perinuclear plane (a) and in a nuclear plane (b). The right-most panels present the enlarged merge images. The nucleus in (a) was detected acquiring the Topro3-signal with increased pin-hole that rendered a non-confocal image. Bars, 5 μm.
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fig02: Immunofluorescence localization of calpain. A. Immunofluorescence images of clones expressing calpain-6-his, calpain-GFP and calpain-2×-flag are presented. The images are representative of the overall population and were obtained using tag-specific primary antibodies and a proper secondary antibody conjugated to Alexa-Fluor (AF) 488. The immunofluorescence distribution in all the clones highlights a region closely associated with the nucleus but not completely merging with the DAPI signal. B. Colocalization of fluorescence signal distributions from both anti-flag antibody and anti-calpain antibody with that of a C-terminal peptide of calpain (#35, Fig. S1). Images a and b show the calpain-2×-flag clone stained with monoclonal anti-flag antibody/goat AF488-conjugated anti-mouse antibody and either rabbit anti-calpain #35/goat AF555-conjugated anti-rabbit or pre-immune serum of the same rabbit/goat AF555-conjugated anti-rabbit. The 3D7 parental strain, stained as in a, is positive with anti-calpain #35, but not with the anti-flag antibody. C. The relative distributions of calpain-2×-flag (green channel) and BiP (red channel) signals were analysed by confocal microscopy. Here two representative images show the signal detected in a perinuclear plane (a) and in a nuclear plane (b). The right-most panels present the enlarged merge images. The nucleus in (a) was detected acquiring the Topro3-signal with increased pin-hole that rendered a non-confocal image. Bars, 5 μm.

Mentions: By immunofluorescence using specific anti-tag antibodies, calpain chimeras (6-his, GFP and 2×-flag) were detected (Fig. 2A). For each we observed a similar fluorescence distribution closely associated with but not completely overlapping that of DAPI-stained nuclei. The fact that all the C-terminal chimeras were detected in similar locations suggests that the localization is independent of the introduced tag and most likely corresponds to the native calpain location. Further, using an anti-calpain peptide antibody, we were able to show that anti-flag and anti-calpain antiserum signals colocalized nicely (Fig. 2B). Colocalization was also observed in the calpain-GFP clone (data not shown). The parental parasite line 3D7 showed a comparable pattern with the anti-calpain antiserum. This confirms that native and tagged calpain have similar locations in the cell. 2×-myc localization was unsuccessful, as tested anti-myc antibodies showed high background above which no significant signal could be detected.


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

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

Immunofluorescence localization of calpain. A. Immunofluorescence images of clones expressing calpain-6-his, calpain-GFP and calpain-2×-flag are presented. The images are representative of the overall population and were obtained using tag-specific primary antibodies and a proper secondary antibody conjugated to Alexa-Fluor (AF) 488. The immunofluorescence distribution in all the clones highlights a region closely associated with the nucleus but not completely merging with the DAPI signal. B. Colocalization of fluorescence signal distributions from both anti-flag antibody and anti-calpain antibody with that of a C-terminal peptide of calpain (#35, Fig. S1). Images a and b show the calpain-2×-flag clone stained with monoclonal anti-flag antibody/goat AF488-conjugated anti-mouse antibody and either rabbit anti-calpain #35/goat AF555-conjugated anti-rabbit or pre-immune serum of the same rabbit/goat AF555-conjugated anti-rabbit. The 3D7 parental strain, stained as in a, is positive with anti-calpain #35, but not with the anti-flag antibody. C. The relative distributions of calpain-2×-flag (green channel) and BiP (red channel) signals were analysed by confocal microscopy. Here two representative images show the signal detected in a perinuclear plane (a) and in a nuclear plane (b). The right-most panels present the enlarged merge images. The nucleus in (a) was detected acquiring the Topro3-signal with increased pin-hole that rendered a non-confocal image. Bars, 5 μm.
© Copyright Policy - open-access
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fig02: Immunofluorescence localization of calpain. A. Immunofluorescence images of clones expressing calpain-6-his, calpain-GFP and calpain-2×-flag are presented. The images are representative of the overall population and were obtained using tag-specific primary antibodies and a proper secondary antibody conjugated to Alexa-Fluor (AF) 488. The immunofluorescence distribution in all the clones highlights a region closely associated with the nucleus but not completely merging with the DAPI signal. B. Colocalization of fluorescence signal distributions from both anti-flag antibody and anti-calpain antibody with that of a C-terminal peptide of calpain (#35, Fig. S1). Images a and b show the calpain-2×-flag clone stained with monoclonal anti-flag antibody/goat AF488-conjugated anti-mouse antibody and either rabbit anti-calpain #35/goat AF555-conjugated anti-rabbit or pre-immune serum of the same rabbit/goat AF555-conjugated anti-rabbit. The 3D7 parental strain, stained as in a, is positive with anti-calpain #35, but not with the anti-flag antibody. C. The relative distributions of calpain-2×-flag (green channel) and BiP (red channel) signals were analysed by confocal microscopy. Here two representative images show the signal detected in a perinuclear plane (a) and in a nuclear plane (b). The right-most panels present the enlarged merge images. The nucleus in (a) was detected acquiring the Topro3-signal with increased pin-hole that rendered a non-confocal image. Bars, 5 μm.
Mentions: By immunofluorescence using specific anti-tag antibodies, calpain chimeras (6-his, GFP and 2×-flag) were detected (Fig. 2A). For each we observed a similar fluorescence distribution closely associated with but not completely overlapping that of DAPI-stained nuclei. The fact that all the C-terminal chimeras were detected in similar locations suggests that the localization is independent of the introduced tag and most likely corresponds to the native calpain location. Further, using an anti-calpain peptide antibody, we were able to show that anti-flag and anti-calpain antiserum signals colocalized nicely (Fig. 2B). Colocalization was also observed in the calpain-GFP clone (data not shown). The parental parasite line 3D7 showed a comparable pattern with the anti-calpain antiserum. This confirms that native and tagged calpain have similar locations in the cell. 2×-myc localization was unsuccessful, as tested anti-myc antibodies showed high background above which no significant signal could be detected.

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