Picking the arginine lock on PQLC2 cycling

A key function of lysosomes is degradation of complex macromolecules, which is followed by export of the constituent metabolites to the cytoplasm. To enable this metabolite export, lysosomes harbor many transporters spanning their limiting membrane. Loss-of-function mutations in lysosomal transporters for lipids, amino acids, and nucleotides are often associated with devastating developmental and neurometabolic diseases, collectively known as lysosomal storage disorders (1, 2).

Along with degradation of macromolecules, lysosomes are also the cellular site of activation for important signaling pathways, such as the master regulator of growth and metabolism, mechanistic target of rapamycin complex 1 (mTORC1) kinase, and the C9orf72-SMCR8-WDR41 (CSW) complex, which plays important but poorly understood roles in neuronal cell homeostasis (3, 4). Emerging evidence indicates that the metabolite-recycling and signaling functions of the lysosome are tightly coupled. Such coupling is mediated, at least in part, by transporter−receptor (“transceptors”) proteins, which possess both solute transport activity and receptor-like signaling activities (5, 6). Lysosomal examples of transceptors include two amino acid transporters, sodium-coupled neutral amino acid transporter 9 (SLC38A9) and proline−glutamine loop repeat-containing protein 2 (PQLC2; pronounced “picklock two”), which have been linked to regulation of mTORC1 and CSW complex, respectively (7–9). An important mechanistic aspect of transporter function is that they must undergo a cyclic conformational change that alternately exposes their solute binding site to either side of the membrane. In the transceptor case, these cyclic conformational rearrangements not only enable solute transport, but they are also key for signaling activity, although how this coupling occurs remains poorly understood. Writing in PNAS, Leray et al. (10) study the dynamics of how specific cationic amino acids (CAA) modulate the gating of the PQLC2 and elucidate a model for the amino acid signaling function of PQLC2.

PQLC2, encoded by the SLC66A1 gene, is a lysosomal CAA transporter belonging to a family of proteins characterized by a seven-helix membrane topology and a highly conserved, duplicated proline−glutamine (PQ) loop motif. Studies with mammalian PQLC2 and yeast vacuolar homologous proteins, Ypq1, Ypq2, and Ypq3, have concluded that PQLC2 selectively exports arginine (Arg), lysine (Lys), and histidine (His) from the lysosomal lumen to the cytosol, and that this activity is strongly dependent on a pH gradient (11). In Caenorhabditis elegans, laat-1 mutants had reduced protein synthesis and had developmental abnormalities, both of which were rescued by external supplementation of Lys and Arg. However, supplementation with CAAs did not reverse the enlarged lysosomes or the defective lysosomal degradation phenotypes associated with laat-1 mutant worms (12). Together, these findings suggest that LAAT-1/PQLC2 is required to maintain cytosolic levels of Lys and Arg by recycling lysosomal CAAs. However, in these earlier studies, the exact transport mechanism for PQLC2 remained unclear.

First, Leray et al. (10) utilized electrophysiology-based techniques in PQLC2-expressing oocytes to determine that CAA transport through PQLC2 is not coupled to protons, and that it operates via a uniport mechanism. Furthermore, although PQLC2 transport activity relies on acidic lysosomal pH, this is a kinetic and not a thermodynamic effect because neither protons nor other ions drive PQLC2 transport. That is, there is no stoichiometric requirement for protons or other ions for PQLC2 transporter function; however, it is likely that an acidic lysosomal pH affects PQLC2 transport rate.

Next, Leray et al. (10) examined the variable effects of the various cationic substrates on the transport current of PQLC2 and found that Arg transport differed significantly from the other CAAs tested (Lys and His). Specifically, PQLC2 oocytes suppressed an outward current upon Arg application, but not upon Lys application, suggesting that luminal Arg is an inward rectifier of PQLC2 current. That is, luminal Arg inhibits PQLC2 uniport activity from the cytosolic to the luminal side. Additionally, cytosolic Arg also decreased current through PQLC2. These findings suggest that Arg, paradoxically, is a PQLC2 substrate in cis and an inhibitor in trans, thereby imposing net transport in the lumen-to-cytosol direction. A kinetic model of Arg-like vs. Lys/His-like transport through PQLC2 clarified that the interaction of Arg with PQLC2 is favored because Arg likely has a higher affinity to the luminal substrate site on PQLC2 and faster closing of the cytosolic gate in the Arg-bound state.

These findings may help to shed light on the important role of Arg in the PQLC2-dependent lysosomal recruitment of the CSW complex (Fig. 1). The CSW complex is recruited to the lysosome through interaction between WDR41 and PQLC2. This interaction is negatively regulated by the availability of cationic amino acids; that is, the CSW complex is maximally recruited to lysosomes when cells are starved of CAAs (9). However, PQLC2 has minimal cytoplasmic domains to mediate amino acid sensing or WDR41 interactions. Previous studies, based on structural predictions and biochemical validation, have shown that WDR41 is recruited to the lysosome by a short peptide motif within a flexible loop that extends from WDR41 and inserts into a large cavity exposed by the cytosol-facing conformation of PQLC2 (6). However, how cationic amino acids control PQLC2 conformational changes has still been an open question.

Fig. 1.

Arg-mediated cycling of PQLC2 under fed and starved conditions. (Top) Under fed states, luminal Arg acts as an inward rectifier for PQLC2, and cytosolic Arg promotes PQLC2 cycling toward the luminal side. Luminal pools of cationic amino acids may be recycled to the cytosol for use in further anabolic pathways and for cellular stress responses. (Bottom) Upon starvation, the lysosome is depolarized, and the C9orf72-SMCR8-WDR42 complex is stabilized on the cytosolic facing conformation of PQLC2. Increased autophagy or refeeding may then increase cytosolic Arg concentrations to resolve starvation and go back to the fed state.

The detailed characterization of Arg modulation of PQLC2 by Leray et al. (10) suggests a model in which cytosolic Arg levels (along with lysosomal polarization) modulate PQLC2 cycling such that, upon starvation, the cytosol-facing conformation may be stabilized. In this case, WDR41 could be recruited to the lysosome to take part in its downstream signaling activities. In such a model, the PQLC2−CSW cycle would be nested within the mTORC1 signaling cycle, whereby starvation may invoke both mTORC1-dependent lysosomal depolarization and autophagy. Increased autophagy, and the consequent rise in luminal CAAs, would ultimately lead to increased cytosolic Arg levels, increased PQLC2 cycling, and a resolution of the starvation phenotypes. However, further experiments will be necessary to validate this idea.

C9orf72 haploinsufficiency is an important genetic cause for two neurodegenerative disorders, amyotrophic lateral sclerosis and frontal temporal dementia (13, 14). The CSW complex, once recruited to the lysosome, was suggested to have GTPase-activating protein (GAP) activities toward ARF1, Rab8a, and Rab11a GTPases based on cryoelectron microscopy structures and GAP activity assays (15–17). It is, therefore, possible that mutations in C9orf72 lead to excess GTP-bound ARF and RAB proteins, which may further lead to the alterations in vesicle trafficking, lysosomal homeostasis, and autophagy that characterize these neurodegenerative diseases. Investigating the details of the PQLC2-dependent recruitment of the CSW complex to lysosomes, and how this recruitment may spatially position the CSW complex relative to its client GTPases, is critical to understand both lysosomal biology and neurodegenerative diseases that arise due to C9orf72 deficiency.

Writing in PNAS, Leray et al. study the dynamics of how specific cationic amino acids (CAA) modulate the gating of the PQLC2 and elucidate a model for the amino acid signaling function of PQLC2.

Outside of the role PQLC2 plays in recruitment of the CSW complex, changes in PQLC2 transport activity may also be associated with defects in how cells cope with stress. In C. elegans, the PQLC2 ortholog, LAAT-1, was required to maintain endoplasmic reticulum homeostasis under stress. This was primarily caused by impaired amino acid transport activity because supplementation with Arg and Lys rescued the LAAT-1 mutant phenotype (18). PQLC2 is also critical in exporting the chemical intermediate formed within lysosomes treated with cysteamine, an important therapeutic for cystinosis, a lysosomal storage disorder that arises from accumulation of cystine within lysosomes due to mutations in the CTNS gene (11). Finally, some reports suggest that PQLC2 may also play a role in mTORC1 signaling, although whether this is through the recruitment of the CSW complex or via the CAA transport function of PQLC2 needs to be characterized further (9).

Altogether, the study by Leray et al. (10) provides a detailed characterization of the transport functions of PQLC2 and, specifically, how Arg modulates this function on both the luminal and the cytosolic sides. These insights will be important in understanding further the role that PQLC2 transceptor plays in health and disease.