The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

Abstract

bMost models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H ؉ -translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H ؉ out of the cell, generating a large electrochemical gradient that supplies energy to H ؉ -coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 m) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex. One of the main features of filamentous fungi is their apical mode of growth. In filamentous fungi, cell wall growth and exocytosis are linked processes that involve the highly polarized traffic of cell wall-building secretory vesicles to apical areas, where they deliver proteins and lipids. One of the most widely accepted models for fungal growth, the vesicle supply center for fungal morphogenesis, postulates a unidirectional traffic of vesicles to the hyphal apex, where they aggregate temporarily at an apical structure, the Spitzenkörper, prior to fusion with the apical plasma membrane (PM) by the process of exocytosis (1). However, some PM proteins, such as the H ϩ -ATPases, essential for hyphal growth, have been previously predicted to be absent or inactive at the hyphal apex (2-4), suggesting the existence of vesicle delivery routes other than the above and independent of the Spitzenkörper that reach nonapical regions of the hyphal PM. H ϩ -ATPases are involved in pumping protons out of the cell, generating a large electrochemical gradient and supplying energy to H ϩ -coupled nutrient uptake systems (5). This electrochemical gradient has been studied in several fungal species by diverse methods, including vibrating probes, microelectrodes, and pH indicators. The results showed that current normally flows inward at the hyphal apical regions and flows outward at distal areas (3, 4, 6-9).PMA1, the H ϩ -ATPase-encoding gene in Saccharomyces cerevisiae (SCRG_01016 [10]), has a single homolog in Neurospora crassa (NCU01680 [11,12]). In S. cerevisiae, Pma1p is one of the most abundant proteins of the cell surface (25 to 50%), while in N. crassa, it constitutes about 5 to 10% of the...

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