Incomplete glycosylation and defective intracellular targeting of mutant solute carrier family 11 member 1 (Slc11a1)

Abstract

Solute carrier family 11 member 1 (Slc11a1, formerly Nramp1) is a highly glycosylated, 12 transmembrane domain protein expressed in macrophages. It resides in the membrane of late endosomes and lysosomes, where it functions as a bivalent cation transporter. Mice susceptible to infection by various intracellular pathogens including Leishmania donovani and Salmonella typhimurium carry a glycine to aspartic acid substitution at position 169 (G169D, Gly(169)-->Asp), within transmembrane domain 4 of Slc11a1. To investigate the molecular pathogenesis of infectious disease susceptibility, we compared the behaviour of heterologously and endogenously expressed wild-type and mutant Slc11a1 by immunofluorescence, immunoelectron microscopy and Western-blot analysis. We found occasional late endosome/lysosome staining of mutant protein using immunoelectron microscopy, but most of the mutant Slc11a1 was retained within the ER (endoplasmic reticulum). Using glycosylation as a marker for protein maturation in two independent heterologous expression systems, we found that most mutant Slc11a1 existed as an ER-dependent, partially glycosylated intermediate species. Correct endosomal targeting of wild-type Slc11a1 continued despite disruption of N-glycosylation sites, indicating that glycosylation did not influence folding or sorting. We propose that the G169D mutation causes localized misfolding of Slc11a1, resulting in its retention in the ER and manifestation of the loss of function phenotype.

Figure 1. Fluorescence localization of EGFP-tagged Slc11a1

Immunofluorescence analysis of the subcellular localization of EGFP-tagged wild-type (A, D) and mutant (G, J) Slc11a1 protein (anti-GFP, green) following transient transfection into RAW264.7 cells. Cells were co-stained (red) either for the LE/Lys marker Lamp1 (B, H) or the early endosome marker EEA1 (E, K). For (D–L), cells were permeabilized with saponin before staining. In the merged panels (C, F, I and L), yellow (C only) indicates co-localization of signal. Scale bar, 10 μm.

Figure 2. Localization of mutant Slc11a1 to the ER

Representative examples of immunofluorescence patterns observed for EGFP-tagged wild-type (A) or mutant (D) Slc11a1 protein (anti-GFP, green) in transiently transfected RAW264.7 cells. Cells were co-stained (red) for the ER marker KDEL (B, E). In the merged panels (C, F), yellow (F only) indicates co-localization of signal. Scale bar, 10 μm.

Figure 3. EM-gold localization of EGFP-tagged Slc11a1

Representative micrographs showing immunoelectron microscopic localization of EGFP-tagged wild-type (A, C and E) and mutant (B, D, F and G) Slc11a1 in the stable macrophage cell lines WT3 and MUT12 respectively. Cryosections were labelled with a rabbit polyclonal antibody to GFP (15 nm gold; A–D), and either a mouse monoclonal to Pdi (10 nm gold; A, B) or a rabbit polyclonal to M6PR (5 nm gold; C, D; arrows). (E–G) Lys were detected by preloading cells with BSA-5 nm gold and labelled for Slc11a1 using the anti-GFP antibody (15 nm gold; arrow heads). Two examples (F, G) are presented for MUT12 because of the low level of staining. Scale bar, 250 nm.

Figure 4. Western-blot analysis of Slc11a1 glycosylation patterns

(A) Extracts (10 μg) from the stable macrophage cell lines WT3 (lane 1) and MUT12 (lane 2) expressing EGFP-tagged wild-type and mutant Slc11a1 respectively, and from untransfected RAW264.7 cells (lane 3) were separated by SDS/PAGE (10% gel). Immunoblotting was performed using an anti-GFP rabbit polyclonal antibody (1:5000) and visualized following a 30 s exposure. Positions of the 72 kDa precursor species and the 97 kDa standard that is flanked by a broad smear of fully glycosylated mature wild-type Slc11a1 are shown on the left. (B) 5 μg of WT3 (lane 1) and 15 μg of MUT12 (lane 2) extracts were immunoblotted as in (A) and visualized following a 10 min exposure to facilitate detection of low levels of mature mutant protein. No staining of untransfected RAW264.7 cells was apparent at this exposure. (C) COS7 cells were transiently transfected with 3×FLAG-tagged wild-type (lane 1) and mutant (lane 2) Slc11a1 and non-recombinant vector (lane 3). Protein was extracted and 10 μg resolved by SDS/PAGE (10% gel), 48 h post-transfection. Immunoblotting was performed using an anti-FLAG monoclonal antibody (1:400). (D) Comparison of EGFP-tagged Slc11a1 expression in RAW and COS7 cells. Extracts (10 μg) from MUT12 (lane 1), WT3 (lane 2) and COS7 cells transiently transfected for 24 h with Slc11a1^wt-EGFP (lane 3), Slc11a1^mut-EGFP (lane 4) and non-recombinant pEGFP-N1 (lane 5) were resolved by SDS/PAGE (10% gel). Immunoblotting was performed as in (A). Positions of the 47 and 54 kDa (C) and 65 and 72 kDa (D) precursor species and the 97 kDa standard band are shown on the left.

Figure 5. Sensitivity to treatment with the glycoamidase PNGase F

(A, B) The protein extract (10 μg) from the stable cell lines WT3 (lanes 1 and 3) and MUT12 (lanes 2 and 4) was denatured and then incubated for 4 h in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 500 units of PNGase F. Samples were resolved by SDS/PAGE (10% gel). Immunoblotting was performed using an anti-GFP rabbit polyclonal antibody (1:5000). After chemiluminescent detection, the blot was exposed for 3 s (A) and 10 s (B). Positions of the 65 and 72 kDa species and the 97 kDa standard are shown on the left. Some degradation of the MUT12 sample is apparent. (C) The extracts (10 μg) from COS7 cells transiently transfected (48 h) with 3×FLAG-tagged wild-type (lanes 1 and 3) or mutant (lanes 2 and 4) Slc11a1 were treated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of PNGase F, as described above. Immunoblotting was performed with an anti-FLAG monoclonal antibody (1:400). Positions of the 47 and 54 kDa species and the 97 kDa standard are shown on the left.

Figure 6. Targeting unaffected by glycosylation state

Subcellular targeting of wild-type Slc11a1 to LE/Lys is unaffected by the glycosylation state. (A) The extracts (10 μg) from parental RAW cells (lane 1), WT3 (lane 2) and CHO-9, the RAW cell clone stably expressing N-glycosylation-deficient Slc11a1 (lane 3), were resolved by SDS/PAGE (10% gel). Immunoblotting was performed using an anti-GFP rabbit polyclonal antibody (1:5000). (B) WT3 cells were cultured for 48 h with tunicamycin: 0 (lane 1), 0.5 μg/ml (lane 2), 0.75 μg/ml (lane 3) and 1.0 μg/ml (lane 4). Protein extracts were prepared and analysed by Western blotting [SDS/PAGE (12% gel), 5–20 μg of protein depending on yield] with anti-GFP rabbit polyclonal antibody (1:5000). Positions of the 65, 72 and 85 kDa proteins and the 97 kDa standard are shown. (C) CHO-9 cells were methanol-fixed and stained for GFP combined with Lamp1. A representative example demonstrating the vesicular staining of N-glycosylation-deficient Slc11a1 clearly co-localizing with Lamp1 is shown. Scale bar, 10 μm.

Figure 7. Localization of endogenous Slc11a1

Localization of endogenous wild-type and mutant Slc11a1 in bone marrow-derived macrophages. Day 10 macrophages from Slc11a1 mutant C57BL/10ScSn versus congenic Slc11a1 wild-type N20, B10.L-Lsh^r mice were activated for 24 h with interferon γ/lipopolysaccharide. After methanol fixation, wild-type (A, C) and mutant (B, D) macrophages were stained with a polyclonal anti-N-terminal Slc11a1 antibody (green), combined with either anti-Lamp1 (A, B) or anti-Pdi (C, D), both shown in red. Merged images are presented with yellow identifying the co-localization of wild-type Slc11a1 with Lamp1 and partial co-localization of mutant Slc11a1 with Pdi. Scale bar, 10 μm.