Regulators of GTP-binding proteins cause morphological changes in the vacuole system of the filamentous fungus, Pisolithus tinctorius.

Abstract

Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving as an alternative to the better-known transport process of vesicular shuttling. In filamentous fungi, tubule formation by vacuoles is particularly pronounced, but little is known of its regulation. Using the hyphae of the basidiomycete Pisolithus tinctorius as our test system, we have investigated the effects of four drugs whose modulation, in animal cells, of the tubule/vesicle equilibrium is believed to be due to the altered activity of a GTP-binding protein (GTP gamma S, GDP beta S, aluminium fluoride, and Brefeldin A). In Pisolithus tinctorius, GTP gamma S, a non-hydrolysable form of GTP, strongly promoted vacuolar tubule formation in the tip cell and next four cells. The effects of GTP gamma S could be antagonised by pre-treatment of hyphae with GDP beta S, a non-phosphorylatable form of GDP. These results support the idea that a GTP-binding protein plays a regulatory role in vacuolar tubule formation. This could be a dynamin-like GTP-ase, since GTP gamma S-stimulated tubule formation has only been reported previously in cases where a dynamin is involved. Treatment with aluminium fluoride stimulated vacuolar tubule formation at a distance from the tip cell, but NaF controls indicated that this was not a GTP-binding-protein specific effect. Brefeldin A antagonised GTP gamma S, and inhibited tubule formation in the tip cell. Given that Brefeldin A also affects the ER and Golgi bodies of Pisolithus tinctorius, as shown previously, it is not clear yet whether the effects of Brefeldin A on the vacuole system are direct or indirect.