The yeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alter pH homeostasis

Abstract

The BTN1 gene product of the yeast Saccharomyces cerevisiae is 39% identical and 59% similar to human CLN3, which is associated with the neurodegenerative disorder Batten disease. Furthermore, btn1-Delta strains have an elevated activity of the plasma membrane H(+)-ATPase due to an abnormally high vacuolar acidity during the early phase of growth. Previously, DNA microarray analysis revealed that btn1-Delta strains compensate for the altered plasma membrane H(+)-ATPase activity and vacuolar pH by elevating the expression of the two genes HSP30 and BTN2. We now show that deletion of either HSP30 or BTN2 in either BTN1(+) or btn1-Delta strains does not alter vacuolar pH but does lead to an increased activity of the vacuolar H(+)-ATPase. Deletion of BTN1, BTN2, or HSP30 does not alter cytosolic pH but diminishes pH buffering capacity and causes poor growth at low pH in a medium containing sorbic acid, a condition known to result in disturbed intracellular pH homeostasis. Btn2p was localized to the cytosol, suggesting a role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase. Increased expression of HSP30 and BTN2 in btn1-Delta strains and diminished growth of btn1-Delta, hsp30-Delta, and btn2-Delta strains at low pH reinforce our view that altered pH homeostasis is the underlying cause of Batten disease.

FIG. 1

The hsp30-Δ and btn2-Δ deletions do not effect response to ANP. The strains were grown for 10 days on YPG medium (A) and YPG medium containing 1.66 mg of ANP/ml (B).

FIG. 2

Diminished growth of btn1-Δ, hsp30-Δ, and btn2-Δ strains at low pH. (A and B) Growth of the ρ⁺ series of strains (a to h) (A) and the ρ⁻ series of strains (i to p) (B) in medium containing 0.2 mM sorbic acid at pH 3.8. (C) Growth of both the ρ⁺ and ρ⁻ series in YPD medium. All strain genotypes are listed in Table 1.

FIG. 3

Btn2p localizes to the cytosol. The btn2-Δ strain containing GFP-Btn2p was grown to mid-exponential phase in a synthetic medium lacking uracil, which allows for normal expression of the BTN2 fusion rather than overexpression utilizing the MET25-promoter. (A) Normal phase-contrast microscopy; (B) fluorescence microscopy; (C) overlay of the phase-contrast and fluorescence images, demonstrating the cytosolic localization of the GFP-Btn2p fusion protein.