# Unusual properties of the lysosomal amino acid transporter PQLC2

Xavier Leray, Yan Li, Anselm A Zdebik, Bruno Gasnier

► To cite this version:

Xavier Leray, Yan Li, Anselm A Zdebik, Bruno Gasnier. Unusual properties of the lysosomal amino acid transporter PQLC2. 60th Annual Meeting of the Biophysical Society, Feb 2016, Los angeles, CA, United States. . hal-04653004

HAL Id: hal-04653004

https://hal.inrae.fr/hal-04653004v1

Submitted on 18 Jul 2024

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# Unusual properties of the lysosomal amino acid transporter PQLC2

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UNIVERSITÉ
PARIS DESCARTES

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Xavier LERAY¹*, Yan LI², Anselm A. ZDEBICK² & Bruno GASNIER¹

(1) Neurophotonics Laboratory, Université Paris Descartes and Centre National de la Recherche Scientifique (CNRS), Paris, France

(2) University College London, United Kingdom

* Xavier Leray is a fellow of the Cystinosis Research Foundation

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CYSTINOSIS
RESEARCH
FOUNDATION

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Lysosomal transporters export the macromolecule building blocks released by acidic lysosomal degradation. In contrast with lysosomal enzymes, they remain poorly studied. The PQ-Loop Containing type-2 (PQLC2) protein is a novel lysosomal transporter that exports cationic amino acids (CAA) from lysosomes (Jezegou et al., PNAS 2012). It belongs to an undercharacterized protein family representing a novel structural fold among membrane transporters. Here, we study its functional properties using two-electrode voltage clamp of Xenopus oocytes expressing PQLC2 at their surface.

Charge-substrate coupling, intracellular acidification and stoichiometry measurements support a uniport mechanism, in contrast with the H+ symport found in most lysosomal transporters. In agreement with this uniport mechanism, PQLC2 activity showed strong voltage dependence. Interestingly, in oocytes previously loaded with CAA, depolarization-induced efflux was silenced when Arg, but not Lys, was present in the bath at a saturating concentration. These last results suggest that PQLC2 shifts from uniport mode in Arg-free medium to an electroneutral antiport mode in the presence of extracellular Arg, but not Lys. This last property suggests a selective role of PQLC2 in regulating lysosomal arginine levels and, possibly, in modulating amino acid sensing by the master protein kinase mTOR.

## 2. PQLC2 is a uniporter

<figure><img src="image_7.png" /><figcaption>B. PQLC2 does not cotransport protons</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C. Measures of stoichiometry confirm the uniport mechanism (for clarification, please read panels 3 and 4 first)</figcaption></figure>

**Experimental procedure :**

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Oocyte is loaded with a prior application of a saturating concentration of Lys. Then reversal potentials (Vrev) are estimated during application of rising concentration of extracellular Lys through I-V measurements.

**Exemples of current traces recorded during voltage jumps :**

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Plotting Vrev as a function of log([Lys]ext) reveals a linear correlation with a slope of approximately 55. This slope indicates that only one Lys (possessing one positive charge) is transported per cycle thanks to Nernst's equation :

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**Same results are obtained with arginine :**

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## 3. PQLC2 transport activity is strongly modulated by the membrane potential

<figure><img src="image_13.png" /><figcaption>B. Current-voltage curves</figcaption></figure>

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## 4. External Arg, but not Lys, shifts PQLC2 from uniport mode to electroneutral antiport mode

### a) Experimental procedure :

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Voltage jumps are done in buffer and with a saturating concentration (20mM) of Arg or Lys. Perfusion is repeated 3 times in different orders.
Two examples of perfusion sequences are represented in A and B.

### b) PQLC2 can reverse :

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Prolonged perfusion of Arg or Lys load the oocyte with these cationic substrates, generating an efflux when returned to the buffer.

### c) Efflux is silenced by external Arg (R), but not Lys (K) :

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The simplest hypotheses for these results are either 1) that Arg inhibits the efflux, or 2) that PQLC2 realizes an electroneutral exchange of one effluxed molecule with one extracellular molecule of arginine.

## 1. Strategy to study PQLC2

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### d) Internal Lys accelerates Arg influx :

Oocytes are first preincubated or not with Lys. Then their membrane potential is depolarized to a positive value (+60mV) and the oocyte is perfused 2min with 3H Arg (0.3mM) and 14C Lys (0.3mM). After washing, oocytes are lysed and accumulated radioactivity is counted.

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Internal Lys efflux allows external Arg to be transported by PQLC2 against the membrane potential. External Arg doesn't inhibit the efflux. The simplest explanation is that in this configuration, PQLC2 shifts from a uniport mode to an electroneutral antiport mode selectively with Arg (not with Lys).

## 5. Conclusion and perspectives
### Proposed model for transport mode

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Uniport
Ping-pong exchange

* Arg would bypass reorientation of empty transporter

* Possible mechanisms: faster transition (depicted); altered transition equilibrium; or altered voltage sensitivity in Arg-bound state

### Future questions to be addressed

* Does this exchange also occur at null or negative potential ?

* Is it symmetrical? (= CAA influx silenced by Arginine) ?

* Does it selectively pick up Arg over other CAAs (histidine, ornithine) ?

* What are the key residues in PQLC2 sequence that contribute to these properties ?

* Biological significance (homeostatic mechanism) ?