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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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Analysis of Pf_calpain expression using C-terminally tagged chimeras. A. Strategy used to create C-terminally tagged calpains by integration at the 3′ end of the endogenous locus. We generated a set of plasmids containing 1.3 kb of sequence from the 3′ end of the Pf_calpain ORF. Downstream of this sequence we placed different tags in frame: 6-his, GFP, 2×-myc and 2×-flag. A human dihydrofolate reductase (hDHFR) cassette was used for positive drug selection. The relative positions of NsiI and SphI restriction sites, used to detect integration, as well as of the probe are indicated. B. Southern blots of restricted genomic DNA. Calpain-6-his, calpain-GFP, calpain-2×-myc and calpain-2×-flag integrant clones gave the bands expected of a single-crossover recombination (white arrows) and plasmid (grey arrows). The asterisks indicate extra bands probably due to concatamerization with plasmid rearrangement, a frequent event in P. falciparum. The 3D7 parental strain shows a single band (black arrows) of the correct size, which is absent in the clones. The difference in size of the smallest DNA fragment reflects the difference in size between GFP and the smaller tags. C. Immunoprecipitated calpain was detected by Western blot analysis. We pulled down calpain using anti-calpain antiserum #19, including as control its pre-immune serum (right panel). For each immunoprecipitation, we used 5 × 108 asynchronous parasites of the parental strain 3D7 (WT) or the calpain-GFP clone (C3). BiP content of each lysate is shown in the panel below. Calpain-GFP was detected by the use of a third antibody, mouse anti-GFP. Pf_calpain is an extremely low-abundance protein that has an apparent molecular weight over 250 kDa (black arrow).
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fig01: Analysis of Pf_calpain expression using C-terminally tagged chimeras. A. Strategy used to create C-terminally tagged calpains by integration at the 3′ end of the endogenous locus. We generated a set of plasmids containing 1.3 kb of sequence from the 3′ end of the Pf_calpain ORF. Downstream of this sequence we placed different tags in frame: 6-his, GFP, 2×-myc and 2×-flag. A human dihydrofolate reductase (hDHFR) cassette was used for positive drug selection. The relative positions of NsiI and SphI restriction sites, used to detect integration, as well as of the probe are indicated. B. Southern blots of restricted genomic DNA. Calpain-6-his, calpain-GFP, calpain-2×-myc and calpain-2×-flag integrant clones gave the bands expected of a single-crossover recombination (white arrows) and plasmid (grey arrows). The asterisks indicate extra bands probably due to concatamerization with plasmid rearrangement, a frequent event in P. falciparum. The 3D7 parental strain shows a single band (black arrows) of the correct size, which is absent in the clones. The difference in size of the smallest DNA fragment reflects the difference in size between GFP and the smaller tags. C. Immunoprecipitated calpain was detected by Western blot analysis. We pulled down calpain using anti-calpain antiserum #19, including as control its pre-immune serum (right panel). For each immunoprecipitation, we used 5 × 108 asynchronous parasites of the parental strain 3D7 (WT) or the calpain-GFP clone (C3). BiP content of each lysate is shown in the panel below. Calpain-GFP was detected by the use of a third antibody, mouse anti-GFP. Pf_calpain is an extremely low-abundance protein that has an apparent molecular weight over 250 kDa (black arrow).

Mentions: The P. falciparum genome contains a single putative calpain gene at the locus MAL13P1.310 (Wu et al., 2003; Croall and Ersfeld, 2007). The sequencing of its cDNA reveals an uninterrupted ORF that encodes a 242 kDa predicted protein (GenBank 432832). To study the enzyme in vivo while altering its physiological regulation as little as possible, we tagged the Pf_calpain C-terminus by homologous single-crossover recombination at the endogenous locus. We constructed a set of vectors containing a hDHFR drug-resistance cassette and 1.3 kb of the Pf_calpain coding region 3′ end. Downstream to this we introduced, in frame, sequences encoding GFP, 6-his, 2×-myc or 2×-flag (Fig. 1A). After two cycles on and off drug, we obtained successful integration of all constructs (Fig. 1B).


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

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

Analysis of Pf_calpain expression using C-terminally tagged chimeras. A. Strategy used to create C-terminally tagged calpains by integration at the 3′ end of the endogenous locus. We generated a set of plasmids containing 1.3 kb of sequence from the 3′ end of the Pf_calpain ORF. Downstream of this sequence we placed different tags in frame: 6-his, GFP, 2×-myc and 2×-flag. A human dihydrofolate reductase (hDHFR) cassette was used for positive drug selection. The relative positions of NsiI and SphI restriction sites, used to detect integration, as well as of the probe are indicated. B. Southern blots of restricted genomic DNA. Calpain-6-his, calpain-GFP, calpain-2×-myc and calpain-2×-flag integrant clones gave the bands expected of a single-crossover recombination (white arrows) and plasmid (grey arrows). The asterisks indicate extra bands probably due to concatamerization with plasmid rearrangement, a frequent event in P. falciparum. The 3D7 parental strain shows a single band (black arrows) of the correct size, which is absent in the clones. The difference in size of the smallest DNA fragment reflects the difference in size between GFP and the smaller tags. C. Immunoprecipitated calpain was detected by Western blot analysis. We pulled down calpain using anti-calpain antiserum #19, including as control its pre-immune serum (right panel). For each immunoprecipitation, we used 5 × 108 asynchronous parasites of the parental strain 3D7 (WT) or the calpain-GFP clone (C3). BiP content of each lysate is shown in the panel below. Calpain-GFP was detected by the use of a third antibody, mouse anti-GFP. Pf_calpain is an extremely low-abundance protein that has an apparent molecular weight over 250 kDa (black arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Analysis of Pf_calpain expression using C-terminally tagged chimeras. A. Strategy used to create C-terminally tagged calpains by integration at the 3′ end of the endogenous locus. We generated a set of plasmids containing 1.3 kb of sequence from the 3′ end of the Pf_calpain ORF. Downstream of this sequence we placed different tags in frame: 6-his, GFP, 2×-myc and 2×-flag. A human dihydrofolate reductase (hDHFR) cassette was used for positive drug selection. The relative positions of NsiI and SphI restriction sites, used to detect integration, as well as of the probe are indicated. B. Southern blots of restricted genomic DNA. Calpain-6-his, calpain-GFP, calpain-2×-myc and calpain-2×-flag integrant clones gave the bands expected of a single-crossover recombination (white arrows) and plasmid (grey arrows). The asterisks indicate extra bands probably due to concatamerization with plasmid rearrangement, a frequent event in P. falciparum. The 3D7 parental strain shows a single band (black arrows) of the correct size, which is absent in the clones. The difference in size of the smallest DNA fragment reflects the difference in size between GFP and the smaller tags. C. Immunoprecipitated calpain was detected by Western blot analysis. We pulled down calpain using anti-calpain antiserum #19, including as control its pre-immune serum (right panel). For each immunoprecipitation, we used 5 × 108 asynchronous parasites of the parental strain 3D7 (WT) or the calpain-GFP clone (C3). BiP content of each lysate is shown in the panel below. Calpain-GFP was detected by the use of a third antibody, mouse anti-GFP. Pf_calpain is an extremely low-abundance protein that has an apparent molecular weight over 250 kDa (black arrow).
Mentions: The P. falciparum genome contains a single putative calpain gene at the locus MAL13P1.310 (Wu et al., 2003; Croall and Ersfeld, 2007). The sequencing of its cDNA reveals an uninterrupted ORF that encodes a 242 kDa predicted protein (GenBank 432832). To study the enzyme in vivo while altering its physiological regulation as little as possible, we tagged the Pf_calpain C-terminus by homologous single-crossover recombination at the endogenous locus. We constructed a set of vectors containing a hDHFR drug-resistance cassette and 1.3 kb of the Pf_calpain coding region 3′ end. Downstream to this we introduced, in frame, sequences encoding GFP, 6-his, 2×-myc or 2×-flag (Fig. 1A). After two cycles on and off drug, we obtained successful integration of all constructs (Fig. 1B).

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