Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase, DnfB

Abstract

SummaryEndocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P-Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkörper from another Type 4 P-type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact-C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4-ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkörper in A. nidulans.

The Golgi apparatus: insights from filamentous fungi

“…PS faces the lumen of the ER, while it is translocated at the cytosolic leaflet by the action of flippases at the trans Golgi (Hankins et al 2015a). The A. nidulans phospholipid translocases (flippases) DnfA (homologue of yeast Dnf1/2) and DnfB (homolog of yeast Drs2) localize at the SPZ and at cytosolic structures, where they co-localize with the late Golgi marker PH OSBP , with DnfB showing a more prominent localization to the latter compartment (Schultzhaus et al 2015). Although the flippases were not formally shown to segregate from early Golgi markers, these data suggested that differences in lipid homeostasis regulation and thus lipid content would further differentiate the early from the late Golgi in A. nidulans.…”

“…Lipid homeostasis at the Golgi has important roles in exocytic cargo sorting (Hankins et al 2015b). Although this is largely an unexplored field in filamentous fungi, the GFP::Lact-C2 reporter binding to phosphatidylserine (PS) recently was used to visualize PS distribution in A. nidulans , Schultzhaus et al 2015. In tip cells, when expressed at relatively high levels, this reporter labels Golgi-like cytosolic structures apart from the SPZ suggesting that PS is enriched on the cytosolic face of these cisternae.…”

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

“…Lipid flippases participate in intracellular membrane organization and secretory pathways [33,45,49,50]. In C. neoformans, previous studies have indicated fundamental roles for putative flippases [26,37–39]; however, functional connections between the primary activity of flippases and their biological roles in fungi have remained unclear.…”

“…In the same organism, a lack of the Neol flippase resulted in defective endocytic and secretory pathways associated with fragmented vacuoles and abnormal processes of membrane fusion [49]. In Aspergillus nidulans, the DnfA flippase is segregated within the Spitzenkorper, which concentrates secretory and endocytic vesicles [50]. Additionally, in Arabidopsis thaliana, deletion of the flippase-coding gene, ALA3, results in the appearance of abnormally large vacuole-like structures [45].…”

Endocytosis and exocytosis in hyphal growth

“…Peñalva et al, additionally, showed that SynA was mislocalized to the PM when its endocytosis was blocked in A. nidulans (Herv as-Aguilar and Peñalva, 2010; Pantazopoulou and Peñalva, 2011). Most recently, Schultzhaus et al showed that a plasma membrane flippase DnfA was recycled through the late Golgi and endocytosed through an NPFxD motif in A. nidulans (Schultzhaus et al, 2015). Fluorescence Recovery After Photobleaching (FRAP) on tip components in C. albicans demonstrated that proteins that localize to the Spitzenk€ orper such as myosin light chain are more transient than proteins that form an apical crescent, such as the exocyst (Jones and Sudbery, 2010).…”