LAAT-1 is the Lysosomal Lysine/Arginine Transporter that Maintains Amino Acid Homeostasis

Defects in exporting hydrolytic degradation products from lysosomes cause lysosomal storage diseases such as cystinosis, which is characterized by intralysosomal accumulation of free cystine because of mutations in the lysosomal cystine transporter gene CTNS (cystinosin) (1-4). The most effective therapeutic agent for cystinosis, cysteamine (an aminothiol), converts lysosomal free cystine to cysteine and the mixed disulfide of cysteine-cysteamine, which is thought to be exported from lysosomes as a lysine analog through a lysine/cationic amino acid transporter (5-7). The molecular identity of the transporter remains unknown. Although biochemically detected, most mammalian lysosomal amino acid transporters have not been molecularly characterized (1).

From a forward genetic screen for C. elegans mutants with increased embryonic cell corpses, we isolated a recessive mutant qx42 that accumulated many refractile corpse-like objects and lysotracker-positive puncta, suggestive of abnormal lysosomes (fig. S1, A to G). Using NUC-1::mCHERRY, which labels lysosomes (8, 9), or lysotracker staining, we found that qx42 lysosomes were on average twice the volume of wild type (1.3 versus 0.5 μm³; Fig. 1, A to F”’ and fig. S1, H to K).

We next examined whether qx42 affected lysosomal cargo degradation. Apoptotic cells are phagocytosed then degraded in lysosomes. Cell death and cell corpse engulfment were normal in qx42 mutants (fig. S2). However, degradation of apoptotic cells in phagolysosomes (indicated by GFP::RAB-7 or NUC-1::mCHERRY) as measured by loss of HIS-24::GFP or H2B::GFP (which label chromatin in all somatic and germ nuclei, including cell corpses, respectively) was severely affected in qx42 mutants, with HIS-24::GFP persisting >4 times as long as in wild type (Fig. 2A and fig. S2, L to O). Yolk lipoprotein is degraded throughout embryogenesis to nourish developing cells (10, 11). In qx42 mutants, intestinal secretion of yolk reporter VIT-2::GFP and uptake by oocytes were normal (fig. S3, A to B’). However, qx42 embryos accumulated significantly more VIT-2::GFP in enlarged puncta, which overlapped with NUC-1::mCHERRY, suggesting defective lysosomal yolk degradation (Fig. 2, B to D and fig. S3, C to H’). Cell surface proteins CAV-1 and RME-2, which are internalized and degraded in wild-type embryos, accumulated in enlarged lysosomes in qx42 embryos (fig. S4) (12). Damaged organelles and protein aggregates are delivered via the autophagy pathway to lysosomes for degradation (13). Autophagy substrates SEPA-1 and T12G3.1 (the C. elegans homolog of mammalian p62) were cleared during embryogenesis in wild type, but persisted in late-stage qx42 mutant embryos and overlapped with NUC-1::mCHERRY, indicating defective autolysosomal degradation (Fig. 2, E to G and fig. S5) (14, 15). Thus, qx42 impairs lysosomal degradation of phagocytic, endocytic and autophagic cargoes.

The gene affected in qx42, Y43H11AL.2, encodes a conserved protein containing seven predicted transmembrane domains and two internal PQ (Proline-Glutamine) loop repeats, characteristic of lysosomal cystine transporters (LCTs) (16) (fig. S6F). Cystinosin, the archetypal LCT family member, is a lysosomal cystine transporter, abnormal function of which causes cystinosis (4). We named the Y43H11AL.2 gene laat-1 (lysosomal amino acid transporter 1) based on similarity with LCT family proteins and cellular functions (see below). qx42 has an A>T mutation in laat-1 that creates a premature stop codon after Asn¹²⁷. Other independently isolated laat-1 mutant alleles also caused enlarged lysosome and persistent cell corpse phenotypes (fig. S1, L to R and S2K). laat-1 was expressed in various cell types in embryos, larvae and adults (fig. S7). GFP or mCHERRY fusion of LAAT-1, which fully rescued qx42 defects (fig. S6, A to E), labeled membranes of NUC-1- or lysotracker-positive structures and overlapped with lysosomal membrane protein CTNS-1, the C. elegans homolog of human cystinosin (17), indicating that LAAT-1 localizes to lysosomal membranes (Fig. 1, G to H” and fig. S7, A to C”). LAAT-1(Δ299-304)::GFP, which lacks the C-terminal dileucine-based lysosomal sorting motif (18), stained plasma membranes instead of lysosomes and failed to rescue laat-1(qx42) mutant phenotypes, indicating that LAAT-1 function depends on its lysosomal localization (fig. S6, A to F and S7, D to E”).

We examined lysosomes purified from C. elegans embryos (fig. S8) and found that loss of CTNS-1 caused cystine accumulation, suggesting that CTNS-1 mediates cystine efflux from lysosomes like human cystinosin (Fig. 3A). In laat-1 mutant lysosomes, cystine levels were normal but lysine and arginine levels were 16 and 8 times as high as wild type, respectively, suggesting that LAAT-1 exports lysine and arginine from lysosomes (Fig. 3A and fig. S9A). Macrophage-like coelomocytes from ctns-1 mutants contained huge granules (>6.5 μm in diameter), which accumulated endocytosed cargo CHERRY and were labeled by lysosomal membrane protein CUP-5 but not endosomal protein RME-8, indicating that they are enlarged lysosomes (19, 20) (Fig. 3, B and C and fig. S9B). Most wild-type and laat-1(qx42) coelomocytes contained small lysosomes (<4.5 μm) or 2-3 bigger ones (4.5 to 6.5 μm) (Fig. 3C). Cysteamine treatment of ctns-1 mutants greatly reduced lysosomal cystine accumulation and almost completely suppressed the enlarged lysosome phenotype (Fig. 3, C and D). In laat-1(qx42)ctns-1(ok813) double mutants, however, cysteamine failed to deplete lysosomal cystine and suppress enlarged lysosomes, which accumulated high levels of cystine and the lysine analog mixed disulfide of cysteine-cysteamine (Fig. 3, C to E). These data strongly suggest that LAAT-1 transports lysine out of lysosomes.

We tested whether LAAT-1 or its human counterpart PQLC2 transported lysine and arginine using a whole cell-based transporter assay (4). Wild-type PQLC2::GFP localized to lysosomes in COS-7 cells, while PQLC2 (ΔLL)::GFP, which lacks the lysosomal sorting motif, associated with plasma membranes, indicating that PQLC2 is a lysosomal membrane protein like LAAT-1 (fig. S6F and fig. S9, C to H”). Expression of plasma membrane-targeted LAAT-1 [LAAT-1(Δ299-304)::GFP] or PQLC2 [PQLC2(ΔLL)::GFP] caused increased uptake of lysine and arginine, which was almost completely abolished when the invariant Pro in the first PQ loop was mutated to Leu (Fig. 3, F and G and fig. S6F). Uptake of histidine but not alanine, glutamic acid, cystine or cysteine was increased in LAAT-1- or PQLC2-expressing cells, suggesting specific transport of cationic amino acids (fig. S10). laat-1 lysosomes did not significantly accumulate histidine, indicating that LAAT-1 is probably not the major histidine transporter in vivo (fig. S9A).

laat-1 mutants were viable but developed slowly (Fig. 4A). External supplements of both lysine and arginine completely rescued retarded embryonic development (Fig. 4B and fig. S11, A and B) but did not reverse the enlarged lysosome or defective yolk degradation phenotypes in laat-1 mutants (fig. S11C). Thus, loss of laat-1 affects lysosomal export of lysine/arginine, which limits their cytoplasmic availability and thereby retards embryonic development. When deprived of amino acids, eukaryotic cells trigger the amino acid response (AAR) pathway through activation of GCN2 protein kinase, leading to repression of global protein synthesis (21). Consistent with this, laat-1 embryos showed reduced protein synthesis, which was efficiently rescued by supplementing lysine and arginine (Fig. 4C and fig. S11D) (22). The AAR pathway is essential for survival during amino acid deprivation (23, 24). gcn-2(ok871) embryos developed normally but died when laat-1 was defective. The synthetic lethality was completely rescued by supplying both lysine and arginine but not glycine (Fig. 4D). Thus, loss of laat-1 limits cytosolic lysine and arginine, causing embryonic lethality when the GCN-2-mediated AAR pathway is impaired (fig. S11E).

We have identified LAAT-1 and its human homolog PQLC2 as the lysosomal lysine/arginine transporter. Cysteamine treatment significantly reduced lysosomal free cystine and efficiently suppressed the enlarged lysosome phenotype in ctns-1(lf) single mutants but not laat-1(lf) ctns-1(lf) double mutants, which accumulated the lysine analog mixed disulfide of cysteine-cysteamine in lysosomes, suggesting that LAAT-1 (and probably PQLC2) may mediate cystine depletion by cysteamine. It is thus important to examine whether loss of PQLC2 affects mammalian lysosome function and causes lysosome-related diseases. Our finding that defective lysosomal export of lysine/arginine leads to retarded embryonic development reveals the role of lysosomal amino acid transporters in maintaining cytosolic amino acid availability during embryonic development, providing insights into the pathogenesis of lysosomal transport disorders.