LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis

Abstract

Defective catabolite export from lysosomes results in lysosomal storage diseases in humans. Mutations in the cystine transporter gene CTNS cause cystinosis, but other lysosomal amino acid transporters are poorly characterized at the molecular level. Here, we identified the Caenorhabditis elegans lysosomal lysine/arginine transporter LAAT-1. Loss of laat-1 caused accumulation of lysine and arginine in enlarged, degradation-defective lysosomes. In mutants of ctns-1 (C. elegans homolog of CTNS), LAAT-1 was required to reduce lysosomal cystine levels and suppress lysosome enlargement by cysteamine, a drug that alleviates cystinosis by converting cystine to a lysine analog. LAAT-1 also maintained availability of cytosolic lysine/arginine during embryogenesis. Thus, LAAT-1 is the lysosomal lysine/arginine transporter, which suggests a molecular explanation for how cysteamine alleviates a lysosomal storage disease.

Figure 1. laat-1 mutants accumulate enlarged lysosomes

(A to F”’) Enlarged lysosomes indicated by NUC-1::mCHERRY [(A) and (B), arrows] or lysotracker red (LTR) [(E) to (F”’), arrowheads] were observed in a laat-1(qx42) embryo (B) or cell [(F) to (F”’)] but not wild type [(A), (E)-(E”’)]. Lysosome volumes are quantified in (C) and (D). Average lysosomal volumes (±SEM, n=100) in different strains are shown in (D). **P<0.0001. (G and H) Fluorescent images of hypodermal (G) or sheath cells (H) in wild-type animals expressing LAAT-1::GFP and NUC-1::mCHERRY. In (A), (B), and (G) to (H”), insets show x4 magnification of lysosomes indicated by yellow arrows. Scale bars: 2 μm in (E), (F); 5 μm in other panels.

Figure 2. laat-1 mutants are defective in lysosomal degradation of various cargoes

(A) Fluorescent images of wild-type and laat-1(qx42) embryos expressing HIS-24::GFP and GFP::RAB-7 at different time points. Arrows indicate phagolysosomes. Quantification is shown in the right panel with average duration (±SEM) shown in parentheses. (B to G) Confocal fluorescent images of wild-type [(B) and (E)] or laat-1(qx42) [(C) and (F)] embryos expressing NUC-1::mCHERRY and VIT-2::GFP [(B) and (C)] or T12G3.1::GFP [(E) and (F)]. Arrows indicate overlapping GFP and mCHERRY; arrowheads indicate non-overlapping GFP. Structures indicated by yellow arrows or arrowheads are magnified x4 in the insets. Quantifications are shown in (D) and (G). At least 10 embryos were scored in each strain. Data are shown as mean±SEM. **P<0.0001. Scale bars: 5 μm.

Figure 3. LAAT-1 is a lysosomal lysine and arginine transporter

(A) The ratio of amino acid concentration in lysosomal versus cytosolic fractions prepared from embryonic lysates was determined and normalized as 1 fold in wild type (y-axis). (B) DIC and fluorescent images of wild-type and ctns-1(ok813) coelomoctyes expressing secreted CHERRY (ssCHERRY) and the lysosomal marker GFP::CUP-5. Lysosomes are labeled by CHERRY and CUP-5 (arrows). Insets show lysosomes indicated by yellow arrows. Scale bars: 5 μm. Quantification is shown in (C). (D and E) Cystine (D) and mixed disulfide of cysteine-cysteamine (E) was determined in purified lysosomal fractions (PLF) after cysteamine treatment and normalized as 1 (fold) in wild type. (F and G) Lysine and arginine uptake was determined in (F) LAAT-1- or (G) PQLC-2-expressing COS-7 cells. Data are shown as mean±SEM. **P<0.0001,*P<0.05; all other points had P>0.05. Data in (A), (D), (E), (F), and (G) are representative of at least 3 independent experiments.

Figure 4. LAAT-1 maintains lysine and arginine availability for normal embryonic development

(A and B) Retarded embryonic development in laat-1 mutants is rescued by external lysine and arginine supplements. At least 88 embryos were examined. (C) Protein synthesis rates determined by fluorescence recovery after photobleaching in wild-type, laat-1(qx42) and laat-1(qx111) embryos expressing P_laat-1mCHERRY with or without externally supplied lysine and arginine. At least 20 embryos were quantified in each strain/treatment. (D) Loss of laat-1 and gcn-2 causes synthetic embryonic lethality. The y-axis indicates the percentage of viable embryos in each strain/treatment. 3 independent experiments were performed with at least 95 embryos examined in each. In panels (C) and (D), data are shown as mean±SEM. **P<0.0001. In panels (B) to (D), lysine (K) and arginine (R) were supplied at 100 mM each and glycine (G) was supplied at 200 mM.