In fungal hyphal cells, intracellular membrane trafficking is constrained by the relatively long intracellular distances and the mode of growth, exclusively by apical extension. Endocytosis plays a key role in hyphal tip growth, which involves the coupling of secretory membrane delivery to the apical region with subapical compensatory endocytosis. However, the identity, dynamics and function of filamentous fungal endosomal compartments remain largely unexplored. Aspergillus nidulans RabA Rab5 localizes to a population of endosomes that show long range bidirectional movement on microtubule (MT) tracks and are labelled with FM4-64 shortly after dye internalization. RabA Rab5 membranes do not overlap with largely static mature endosomes/vacuoles. Impaired delivery of dynein to the MT plus ends or downregulation of cytoplasmic dynein using the dynein heavy chain nudA1 ts mutation results in accumulation of RabA Rab5 endosomal membranes in an abnormal NudA1 compartment at the tip, strongly supporting the existence in A. nidulans hyphal tips of a dynein loading region. We show that the SynA synaptobrevin endocytic recycling cargo traffics through this region, which strongly supports the contention that polarized hyphal growth involves the association of endocytic recycling with the plus ends of MTs located at the tip, near the endocytic internalization collar.

Of the two Aspergillus early endosomal Rab5 paralogues, RabB recruits, in its GTP conformation, Vps19, Vps45, and Vps34, and the CORVET complex and couples acquisition of PI(3)P degradative identity with the long-distance movement of early endosomes. RabA also recruits CORVET, albeit less efficiently. The simultaneous loss of RabA and RabB is lethal.

In yeast, the TRAPP complexes activate Rab1 with TRAPPII also activating Rab11, but less is known about the two TRAPPs in metazoans. Riedel et al. show that in Drosophila melanogaster, TRAPPIII is an essential Rab1 activator, and TRAPPII activates Rab1 and Rab11 and becomes essential when an unrelated Rab11 activator is deleted.

The seven-transmembrane receptor PalH and its coupled, positive-acting arrestin-like protein PalF are key components of a molecular sensor that in Aspergillus nidulans and other ascomycete fungi mediates activation of an intracellular signaling cascade by alkaline ambient pH. PalF is ubiquitinated in an alkaline pH-and PalH-dependent manner. We show here that PalF assists the plasma membrane localization of PalH and that PalF overexpression slightly hypersensitizes the pathway to alkaline pH but does not bypass the need for the ambient pH signal receptor in signaling. In contrast, covalent attachment of Ub to PalF activates the signaling pathway under acidic pH conditions in which the pathway is normally inactive, demonstrating a positive role for ubiquitination. We further show that PalF acts upstream of, or in concert with, the Bro1 domain-containing pH signaling protein PalC, which is normally recruited to cortical structures likely to represent active pH signaling foci under neutral/ alkaline pH conditions. In agreement with its pathwayactivating consequences, expression of PalF-Ub also promotes PalC cortical recruitment under acidic conditions. Notably, our data establish that expression of PalF-Ub, at approximately physiological levels, in a null palH background leads to a considerable degree of signaling even in the complete absence of the receptor. Thus PalF ubiquitination is a key, perhaps the sole, molecular trigger required for transmitting the alkaline pH signal to the downstream elements of the pathway.