Uptake and compartmentalisation of fluorescent probes by Pisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells of Pisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the novel fluorescein-substitute Oregon Green 488 carboxylic acid diacetate, and the thiol-reactive Cell Tracker reagents 7-amino-4-chloro-methylcoumarin and 5-chloromethylfluorescein diacetate, are taken up by these hyphae and their fluorescent products accumulate in the vacuole system. Accumulation of the fluorescent products of both 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate in the vacuole system is inhibited by the anion transport inhibitor probenecid and instead these fluorochromes remain in the cytoplasm. These results suggest that the membrane-permeant esters 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate are first hydrolysed in the cytoplasm and that their fluorescent products are subsequently sequestered across the tonoplast via an anion transport mechanism. Such an anion transport mechanism has been hitherto unrecognised in fungi and may serve to detoxify the fungal cytoplasm by the removal of naturally-occurring unwanted anions. Probenecid-inhibitable organic anion transporters are also located at the limiting membrane of the animal endosomal/lysosomal system and at the tonoplast of higher plants. Our results further support the idea that the tubular vacuole system in P. tinctorius is similar to animal endosomal/lysosomal and plant vacuole systems.