A role in vacuolar arginine transport for yeast Btn1p and for human CLN3, the protein defective in Batten disease

Abstract

In Saccharomyces cerevisiae, transport of arginine into the vacuole has previously been shown to be facilitated by a putative H+/arginine antiport. We confirm that transport of arginine into isolated yeast vacuoles requires ATP and we demonstrate a requirement for a functional vacuolar H+-ATPase. We previously reported that deletion of BTN1 (btn1-delta), an ortholog of the human Batten disease gene CLN3, resulted in a decrease in vacuolar pH during early growth. We report that this altered vacuolar pH in btn1-delta strains underlies a lack of arginine transport into the vacuole, which results in a depletion of endogenous vacuolar arginine levels. This arginine transport defect in btn1-delta is complemented by expression of either BTN1 or the human CLN3 gene and strongly suggests a function for transport of, or regulation of the transport of, basic amino acids into the vacuole or lysosome for yeast Btn1p, and human CLN3 protein, respectively. We propose that defective transport at the lysosomal membrane caused by an absence of functional CLN3 is the primary biochemical defect that results in Batten disease.

Fig. 1.

Isolated vacuoles from yeast display ATP-dependent and vacuolar H⁺-ATPase-dependent transport of arginine into the vacuole. (A) Uptake of [¹⁴C]arginine into isolated vacuoles from wild-type yeast (B-11718) in the presence (•) and absence (○) of ATP. Results shown are typical of those obtained from six independently derived uptake experiments. (B) Lineweaver–Burk plot with 95% confidence limits, showing first-order kinetic uptake, K_m = 0.02 mM, of [¹⁴C]arginine into isolated vacuoles from wild-type yeast, B-11718. (C) Rate of uptake of [¹⁴C]arginine into isolated vacuoles from wild-type (WT) yeast, B-11718; yeast lacking the CUP5-encoded V₀ subunit of the vacuolar H⁺-ATPase (cup5-Δ), B-12074; yeast lacking the VMA13-encoded V₁ subunit of the vacuolar H⁺-ATPase (vma13-Δ), B-13046; and yeast lacking Btn1p (btn1-Δ), B-13048. Results were obtained from four independently derived preparations of vacuoles from each strain. All isolated vacuoles shown were prepared from strains grown on YPD.

Fig. 2.

Isolated vacuoles from btn1-Δ cells display a lack of transport into the vacuole that is complemented by yeast BTN1 and overexpression of human CLN3. (A) Rate of uptake of [¹⁴C]arginine into isolated vacuoles from btn1-Δ, B-13048; btn1-Δ with plasmid-borne BTN1, B-13048 plus DAP142 (pAB1052-BTN1), Y002; btn1-Δ with plasmid vector only, B-13048 plus pAB1052, Y001; and wild-type, B-11718. Results were obtained from four independently derived preparations of vacuoles from each strain. Strains were grown on synthetic complete medium minus uracil, except for B-13048 and B-11718, which had uracil added to the medium to facilitate growth. (B) Rate of uptake of [¹⁴C]arginine into isolated vacuoles from btn1-Δ, B-13048; btn1-Δ with plasmid-borne human CLN3, B-13048 plus DAP (pAB1052-CLN3), Y003; and btn1-Δ with plasmid vector only, B-13048 plus pAB1052, Y001. Results were obtained from four independently derived preparations of vacuoles from each strain. Strains were grown on synthetic complete medium lacking uracil, followed by washing and resuspension into synthetic complete galactose medium lacking uracil for 4 hr for induction purposes. Note that, for B-13048, uracil was added to the medium to facilitate growth.