Lysosomes are acidic organelles which need to export substrates generated by hydrolysing digested carbohydrates, lipids and proteins. Cystinosin is the prototypic member of a family of heptahelical membrane proteins featuring a dual PQ loop motif. Recent work has identified an aspartate coupling the transport of protons to the translocation of uncharged cystine. We now targeted another member of this family to the plasma membrane by disrupting a dileucine lysosomal sorting motif and show that PQLC2 transports basic amino acids including arginine and histidine with millimolar affinity. We investigated whether this protein also co-transports protons using pH measurements in mammalian cels as well as Fluorocyte, a technique which combines semiquantitative pH measurements and TEVC in oocytes. Histidine-induced inward currents were ~3fold larger at pH 6 than at 7.5 in the bath, whereas arginine-induced currents were 4fold larger. Control oocytes showed negligible inward currents in response to both amino acids. Interestingly, a robust intracellular acidification was observed only with histidine, but not arginine. Fig. 1 shows an example experiment where transport was increased by switching the clamp protocol from pulses to +20 mV to pulses to -100 mV. We conclude that deprotonation of histidine, which can occur in the near-neutral cytosolic pH releases protons, whereas the more alkaline pK of arginine prevents significant deprotonation once these amino acids have been translocated into the cytosol. Sequence comparison with cystinosin showed that D305, necessary for proton transport by cystinosin, is lacking in PQLC2, which could explain the absence of proton coupling in this transporter.

Figure 1