Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2

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Abstract

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P5B ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor–Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor–Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.

Introduction

Kufor–Rakeb syndrome (KRS) is a rare form of juvenile Parkinsonism that follows autosomal recessive inheritance, first described in 1994 by al-Din et al. Manifesting between 7 and 24 years of age, these patients present with juvenile-onset Parkinsonian symptoms attributed to pallido-pyrimidal syndrome (PPS), including bradykinesia, paraparesis, stooped posture and hyperreflexia [12], [24], [34]. Kufor–Rakeb patients also have distinct symptoms including widespread neurodegeneration resulting in dementia and upgaze paresis, yet lack the intention tremor typical to Parkinsonism disorders [3], [12], [22], [34], [45]. The causative gene associated with KRS is ATP13A2. ATP13A2 encodes an 1180 amino acid P-type ATPase, specifically of the P5 subfamily, that localizes to the lysosome [5], [26], [37]. There are several mutations identified with various forms of KRS [14], [30], [37].

Ypk9p (YOR291w) is the Saccharomyces cerevisiae homologue to human ATP13A2 based on amino acid sequence alignment. Ypk9p is a 1472 amino acid P5 ATPase and has 58% similarity and 38% identity to ATP13A2. Recently, Ypk9p was shown to suppress α-synuclein and manganese toxicity in yeast, revealing a connection between the yeast gene and PD genetic and environmental risk factors [18]. We demonstrate that deletion of YPK9, ypk9-Δ, results in sensitivity to cadmium, manganese, nickel and selenium. Further studies of the yeast protein may help to elucidate the function of ATP13A2 and uncover underlying defects of Kufor–Rakeb syndrome.

Section snippets

Materials and methods

Strains and plasmid construction. The parental (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) and single deletion strains used in this study were purchased from Open BioSystems. YPK9-GST in pEGH was purchased from Open Biosystems and transformed into ypk9-Δ using standard lithium acetate transformation protocol [23], [40]. ATP13A2 was amplified from human cDNA and subcloned into pcDNA3.1 (Invitrogen) and ligated into pYeura3 (Clontech) and transformed into YPK9+ and ypk9-Δ strains. YPK9-GFP was purchased

Identification of YPK9

YOR291w is the yeast homolog to human ATP13A2 (58% similarity and 38% identity). ATP13A2 and Ypk9p both contain a PPALP sequence at the proposed ion binding site, classifying them as P5 ATPases of the P5B subfamily [33]. Spf1p, the other yeast P-type ATPase, is classified as a P5A ATPase and is presumed to have different ion specificities from Ypk9p based on the presence of two negative charges in place of the hydrophobic residue in P5B ATPases [33]. There appears to be some functional overlap

Discussion

YPK9 is the yeast homolog of human gene ATP13A2, which is mutated in Kufor–Rakeb syndrome patients. Our results suggest that Ypk9p could play a role in resistance to cadmium, manganese, nickel and selenium in the vacuole. However, nickel and selenium resistance may be influenced by the status of histidine metabolism. The sensitivity of ypk9-Δ to these metals may result from the inability of the cells to sequester the surplus of metals in the vacuole, thereby increasing cytosolic concentrations

Acknowledgments

This work was supported in part by NIH R01 NS36610 and P30 ES01247.

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