The role of the Parkinson's disease gene PARK9 in essential cellular pathways and the manganese homeostasis network in yeast

Abstract

YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.

Figure 1. Yeast genetic screens provide insight into the manganese homeostasis network.

A, Screen for manganese resistant deletion strains. A representative plate is shown (each plate contains 384 different yeast deletion strains pinned in duplicate, 768 total colonies). Left, control plate (0 mM Mn²⁺); right, plate containing 25 mM Mn²⁺. The red arrow points to a manganese resistant strain, growing better than the other strains in the presence of a toxic concentration of Mn²⁺. B, Manganese resistance in the mam3Δ strain is independent of Ypk9 function. A representative spotting assay showing that resistance to Mn²⁺ in the mam3Δ strain is independent of Ypk9. Five-fold serial dilutions of WT, ypk9Δ, mam3Δ and mam3Δ ypk9Δ were spotted on SD plates containing different Mn²⁺ concentrations. C, Functional categories of genes enriched as manganese sensitive hits in the Mn²⁺ tolerance screen compared to the yeast genome. D, Functional categories of genes enriched as manganese resistant hits in the Mn²⁺ tolerance screen compared to the yeast genome.