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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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Interaction of HC1 and HC3 regions in reporter targeting. A and B. Fluorescence of live P. falciparum (left; bars, 5 μm) and fixed human 293T cells (right; bar, 1 μm). As in Figs 5 and 6, schematics of the constructs and mutations are shown and images are labelled at the top. Chimeras shown have the short HC1 version (Nt) (A) or long one (NtL) (B). C. Analysis of expressed constructs in P. falciparum. As in Fig. 6, chimeras, immunoprecipitated from stably transfected parasites, were analysed by Western blot using rabbit anti-GFP antibody (top) and, after stripping of the membrane, rabbit-anti-calpain antiserum #17 (bottom). D and E. Analysis of constructs in mammalian cells. 293T cells were transfected with plasmids encoding each of the chimeras as in (C). The cells were grown in normal (D), or labelling media (E). In (D), cell lysates were analysed by Western blotting using anti-GFP (top) to monitor expression levels or anti-calpain antiserum #17 (bottom). E. Alternatively, lysates were immunoprecipitated with anti-GFP antibody and fluorography was performed. F. Cellular fractionation analysis of Ntl-YFP-NLS and its two HC1 mutants, G2A and C3A. The distribution of the small chimeras (panel a) and Plasmepsin V (PMV), a membrane marker (panel b) in the total lysate (1/2 vol.), soluble (1 vol.) and membrane fractions (1 vol.) is shown.
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fig07: Interaction of HC1 and HC3 regions in reporter targeting. A and B. Fluorescence of live P. falciparum (left; bars, 5 μm) and fixed human 293T cells (right; bar, 1 μm). As in Figs 5 and 6, schematics of the constructs and mutations are shown and images are labelled at the top. Chimeras shown have the short HC1 version (Nt) (A) or long one (NtL) (B). C. Analysis of expressed constructs in P. falciparum. As in Fig. 6, chimeras, immunoprecipitated from stably transfected parasites, were analysed by Western blot using rabbit anti-GFP antibody (top) and, after stripping of the membrane, rabbit-anti-calpain antiserum #17 (bottom). D and E. Analysis of constructs in mammalian cells. 293T cells were transfected with plasmids encoding each of the chimeras as in (C). The cells were grown in normal (D), or labelling media (E). In (D), cell lysates were analysed by Western blotting using anti-GFP (top) to monitor expression levels or anti-calpain antiserum #17 (bottom). E. Alternatively, lysates were immunoprecipitated with anti-GFP antibody and fluorography was performed. F. Cellular fractionation analysis of Ntl-YFP-NLS and its two HC1 mutants, G2A and C3A. The distribution of the small chimeras (panel a) and Plasmepsin V (PMV), a membrane marker (panel b) in the total lysate (1/2 vol.), soluble (1 vol.) and membrane fractions (1 vol.) is shown.

Mentions: No palmitate labelling was detected in G2A mutants (Fig. 6D panel b), as in this type of motif palmitoylation depends on prior myristoylation (Resh, 1999). However, when Cys3 was mutated to Ala, palmitate labelling of the chimeras was not abolished but only reduced, indicating the presence of a second palmitoylation site (see below). These results suggested that HC1 contains a functional acylation motif that anchors the protein to membranes. Further evidence is provided below (Figs 7 and 9).


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

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

Interaction of HC1 and HC3 regions in reporter targeting. A and B. Fluorescence of live P. falciparum (left; bars, 5 μm) and fixed human 293T cells (right; bar, 1 μm). As in Figs 5 and 6, schematics of the constructs and mutations are shown and images are labelled at the top. Chimeras shown have the short HC1 version (Nt) (A) or long one (NtL) (B). C. Analysis of expressed constructs in P. falciparum. As in Fig. 6, chimeras, immunoprecipitated from stably transfected parasites, were analysed by Western blot using rabbit anti-GFP antibody (top) and, after stripping of the membrane, rabbit-anti-calpain antiserum #17 (bottom). D and E. Analysis of constructs in mammalian cells. 293T cells were transfected with plasmids encoding each of the chimeras as in (C). The cells were grown in normal (D), or labelling media (E). In (D), cell lysates were analysed by Western blotting using anti-GFP (top) to monitor expression levels or anti-calpain antiserum #17 (bottom). E. Alternatively, lysates were immunoprecipitated with anti-GFP antibody and fluorography was performed. F. Cellular fractionation analysis of Ntl-YFP-NLS and its two HC1 mutants, G2A and C3A. The distribution of the small chimeras (panel a) and Plasmepsin V (PMV), a membrane marker (panel b) in the total lysate (1/2 vol.), soluble (1 vol.) and membrane fractions (1 vol.) is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig07: Interaction of HC1 and HC3 regions in reporter targeting. A and B. Fluorescence of live P. falciparum (left; bars, 5 μm) and fixed human 293T cells (right; bar, 1 μm). As in Figs 5 and 6, schematics of the constructs and mutations are shown and images are labelled at the top. Chimeras shown have the short HC1 version (Nt) (A) or long one (NtL) (B). C. Analysis of expressed constructs in P. falciparum. As in Fig. 6, chimeras, immunoprecipitated from stably transfected parasites, were analysed by Western blot using rabbit anti-GFP antibody (top) and, after stripping of the membrane, rabbit-anti-calpain antiserum #17 (bottom). D and E. Analysis of constructs in mammalian cells. 293T cells were transfected with plasmids encoding each of the chimeras as in (C). The cells were grown in normal (D), or labelling media (E). In (D), cell lysates were analysed by Western blotting using anti-GFP (top) to monitor expression levels or anti-calpain antiserum #17 (bottom). E. Alternatively, lysates were immunoprecipitated with anti-GFP antibody and fluorography was performed. F. Cellular fractionation analysis of Ntl-YFP-NLS and its two HC1 mutants, G2A and C3A. The distribution of the small chimeras (panel a) and Plasmepsin V (PMV), a membrane marker (panel b) in the total lysate (1/2 vol.), soluble (1 vol.) and membrane fractions (1 vol.) is shown.
Mentions: No palmitate labelling was detected in G2A mutants (Fig. 6D panel b), as in this type of motif palmitoylation depends on prior myristoylation (Resh, 1999). However, when Cys3 was mutated to Ala, palmitate labelling of the chimeras was not abolished but only reduced, indicating the presence of a second palmitoylation site (see below). These results suggested that HC1 contains a functional acylation motif that anchors the protein to membranes. Further evidence is provided below (Figs 7 and 9).

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