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Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.

Gruenheid S, Pinner E, Desjardins M, Gros P - J. Exp. Med. (1997)

Bottom Line: In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment.After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5.The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

ABSTRACT
The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Bcg, Ity, Lsh) controls the innate resistance or susceptibility of mice to infection with a group of unrelated intracellular parasites which includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expressed exclusively in professional phagocytes and encodes an integral membrane protein that shares structural characteristics with ion channels and transporters. Its function and mechanism of action remain unknown. The intracellular localization of the Nramp1 protein was analyzed in control 129/sv and mutant Nramp1-/- macrophages by immunofluorescence and confocal microscopy and by biochemical fractionation. In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment. Double immunofluorescence studies and direct purification of latex bead-containing phagosomes demonstrated that upon phagocytosis, Nramp1 is recruited to the membrane of the phagosome and remains associated with this structure during its maturation to phagolysosome. After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5. The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.

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Subcellular localization of the Nramp1 protein in  macrophages. Peritoneal macrophages from normal 129/sv  mice (A) and from 129/sv  Nramp1−/− mutants (B) were  harvested by peritoneal lavage,  cultured for 48 h, and analyzed  by indirect immunofluorescence  with an anti-Nramp1 rabbit  polyclonal antibody (GST-35C,  at 1:50 dilution) raised against  the 35 COOH-terminal residues of Nramp1. The secondary  antibody was a goat anti–rabbit  antiserum conjugated to Texas  red (1:200 dilution). Cells in A  and B were processed identically,  and equal exposure times were  used for photography.
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Figure 1: Subcellular localization of the Nramp1 protein in macrophages. Peritoneal macrophages from normal 129/sv mice (A) and from 129/sv Nramp1−/− mutants (B) were harvested by peritoneal lavage, cultured for 48 h, and analyzed by indirect immunofluorescence with an anti-Nramp1 rabbit polyclonal antibody (GST-35C, at 1:50 dilution) raised against the 35 COOH-terminal residues of Nramp1. The secondary antibody was a goat anti–rabbit antiserum conjugated to Texas red (1:200 dilution). Cells in A and B were processed identically, and equal exposure times were used for photography.

Mentions: We have previously reported the production of a series of specific rabbit anti-Nramp1 polyclonal antisera (24), including the GST-35C serum raised against a protein comprising the COOH-terminal 35 residues of Nramp1 fused to GST. In immunoprecipitation and immunoblotting experiments using extracts from macrophages, this serum identified Nramp1 as an integral membrane phosphoglycoprotein of 90–95 kD (24), in agreement with structural and functional features predicted from the sequence of Nramp1 cDNA (12). GST-35C was used to localize the Nramp1 protein in macrophages by indirect immunofluorescence (Fig. 1). In 129/sv macrophages (Fig. 1 A), we observed a strong intracellular vesicular staining pattern that was intense in the perinuclear region but also extended throughout the length of the long cellular processes. This staining was specific and absent in macrophages from control Nramp1−/− mutants (Fig. 1 B). A similar staining pattern was observed using an unrelated anti-Nramp1 antiserum directed against the 53 NH2-terminal residues of the protein (data not shown). Finally, we did not detect any Nramp1 staining associated with either the plasma membrane or the nuclear membrane, indicating that Nramp 1 expression is restricted to a subcellular, probably membranous, compartment.


Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.

Gruenheid S, Pinner E, Desjardins M, Gros P - J. Exp. Med. (1997)

Subcellular localization of the Nramp1 protein in  macrophages. Peritoneal macrophages from normal 129/sv  mice (A) and from 129/sv  Nramp1−/− mutants (B) were  harvested by peritoneal lavage,  cultured for 48 h, and analyzed  by indirect immunofluorescence  with an anti-Nramp1 rabbit  polyclonal antibody (GST-35C,  at 1:50 dilution) raised against  the 35 COOH-terminal residues of Nramp1. The secondary  antibody was a goat anti–rabbit  antiserum conjugated to Texas  red (1:200 dilution). Cells in A  and B were processed identically,  and equal exposure times were  used for photography.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196151&req=5

Figure 1: Subcellular localization of the Nramp1 protein in macrophages. Peritoneal macrophages from normal 129/sv mice (A) and from 129/sv Nramp1−/− mutants (B) were harvested by peritoneal lavage, cultured for 48 h, and analyzed by indirect immunofluorescence with an anti-Nramp1 rabbit polyclonal antibody (GST-35C, at 1:50 dilution) raised against the 35 COOH-terminal residues of Nramp1. The secondary antibody was a goat anti–rabbit antiserum conjugated to Texas red (1:200 dilution). Cells in A and B were processed identically, and equal exposure times were used for photography.
Mentions: We have previously reported the production of a series of specific rabbit anti-Nramp1 polyclonal antisera (24), including the GST-35C serum raised against a protein comprising the COOH-terminal 35 residues of Nramp1 fused to GST. In immunoprecipitation and immunoblotting experiments using extracts from macrophages, this serum identified Nramp1 as an integral membrane phosphoglycoprotein of 90–95 kD (24), in agreement with structural and functional features predicted from the sequence of Nramp1 cDNA (12). GST-35C was used to localize the Nramp1 protein in macrophages by indirect immunofluorescence (Fig. 1). In 129/sv macrophages (Fig. 1 A), we observed a strong intracellular vesicular staining pattern that was intense in the perinuclear region but also extended throughout the length of the long cellular processes. This staining was specific and absent in macrophages from control Nramp1−/− mutants (Fig. 1 B). A similar staining pattern was observed using an unrelated anti-Nramp1 antiserum directed against the 53 NH2-terminal residues of the protein (data not shown). Finally, we did not detect any Nramp1 staining associated with either the plasma membrane or the nuclear membrane, indicating that Nramp 1 expression is restricted to a subcellular, probably membranous, compartment.

Bottom Line: In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment.After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5.The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

ABSTRACT
The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Bcg, Ity, Lsh) controls the innate resistance or susceptibility of mice to infection with a group of unrelated intracellular parasites which includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expressed exclusively in professional phagocytes and encodes an integral membrane protein that shares structural characteristics with ion channels and transporters. Its function and mechanism of action remain unknown. The intracellular localization of the Nramp1 protein was analyzed in control 129/sv and mutant Nramp1-/- macrophages by immunofluorescence and confocal microscopy and by biochemical fractionation. In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment. Double immunofluorescence studies and direct purification of latex bead-containing phagosomes demonstrated that upon phagocytosis, Nramp1 is recruited to the membrane of the phagosome and remains associated with this structure during its maturation to phagolysosome. After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5. The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.

Show MeSH
Related in: MedlinePlus