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, Gly169→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.