Mechanistic Insights into Manganese Induced Down - Regulation of the Cis Golgi Glycoprotein GPP130

During invasion of host cells the Shigella bacterial toxin sorts away from degradative lysosomes and traffics to the Golgi complex by binding the cycling transmembrane protein GPP130 as GPP130 returns from endosomes to the Golgi. Remarkably, an increase in Golgi manganese (Mn) alters the trafficking of GPP130 causing its degradation in lysosomes thereby protecting cells against lethal doses of purified toxin. Mn-induced down-regulation of GPP130 is thus an important paradigm for therapy against the incurable Shiga toxicosis as well as metal-regulated protein sorting in the Golgi. The experiments herein reveal significant aspects of the mechanism. We identified a transferable trafficking determinant within the GPP130 lumenal stem domain that bound Mn and observed that Mn binding induced its oligomerization in the Golgi. Significantly, alanine substitutions that blocked Mn binding abrogated both oligomerization and GPP130 sorting to lysosomes. GPP130 oligomers engaged the canonical Golgi-lysosomal trafficking machinery, as GPP130 exit from the Golgi required the sorting adaptor GGA1 and the clathrin vesicle coat complex. Surprisingly, oligomerization was sufficient since GPP130 redistributed to lysosomes in the absence of Mn by forced aggregation using a drug-controlled selfinteracting version of the FKBP domain. Further, neither the cytoplasmic domain nor residues in the lumenal stem domain critical for normal GPP130 cycling were required for this redistribution. These observations suggested that higher order oligomerization might generally cause lysosomal targeting of Golgi membrane proteins. Indeed, induced oligomerization of two other unrelated Golgi proteins caused their redistribution to endosome/lysosome-like punctae and subsequent degradation. The ER-based unfolded protein response was not activated during this process. Altogether, these results show that Mn binds and oligomerizes GPP130 targeting it to what may be a pre-existing quality control pathway in the Golgi where aggregated proteins are degraded in lysosomes.