Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth

Polarized growth is a biological process observed for many different eukaryotic cell systems, including pollen tube formation (50), neuronal cell development (7), and fungal hyphal growth (5). Filamentous fungi are characterized by extensive polarized growth throughout most of their life cycles. This unique reliance on hyphal morphogenesis makes filamentous fungi an attractive system to study polarized growth. Invasive hyphal growth is an important strategy for animal and plant pathogenic fungi to infect and penetrate their hosts. Therefore, understanding hyphal growth will be critical to the development of therapeutic or antifungal agents. Girbardt reported the existence of the Spitzenkorper (apical body) at growing hyphae and proposed that the Spitzenkorper coincided with the polarized growth site (18). The Spitzenkorper consists of two populations of vesicles, apical vesicles (79 to 90 nm in diameter) and microvesicles (30 to 40 nm in diameter). Microvesicles possess chitin synthesis activity and have been called “chitosomes” (4, 6). The postulated vesicle supply center thought to be necessary to explain hyphal tip growth (19, 20) may correspond with the Spitzenkorper. This has driven the hypothesis that vesicle assembly and trafficking play key roles in polarized growth in filamentous growth. ADP ribosylation factors (Arfs) are small GTPase proteins that function in vesicle assembly and trafficking (34, 36). In Saccharomyces cerevisiae, Arf3 is involved in polarity establishment during budding and an arf3 mutant shows a random budding phenotype (16, 22). The Arf3 protein localizes to cell membranes and predominantly to sites of polarized growth (22). Arf3 also genetically interacts with actin cable and actin patch components, including profilin (Pfy1), Arp2/3 complex protein (Las17p and Vrp1), formins (Bni1 and Bnr1), Arf-GTP exchange factor (Gea1), and actin-organizing proteins (Syp1 and Bud6) (30). Another function of Arf3p in yeast is in membrane trafficking during endocytosis (9, 10). ARF6, the human homolog of S. cerevisiae Arf3p (ScArf3p), is also involved in endocytosis, cytokinesis, and actin cytoskeleton organization (reviewed in reference 17). Shaw et al. reported that a temperature-sensitive N-myristoyl transferase (NMT) mutant displayed a polarity maintenance defect in Aspergillus nidulans (43). Myristate from myristoyl-coenzyme A (CoA) is covalently attached to the secondary glycine of target proteins by NMT increasing in hydrophobicity (24). Myristoylated protein more readily associates with membranes or takes part in hydrophobic protein-protein interactions (24, 41). Many Arfs, including ScArf3p, are N myristoylated (2, 35). We hypothesized that Arf proteins may be the direct connection between N myristoylation and polarized cell morphogenesis in filamentous fungi. Indeed, in S. cerevisiae, N-myristoylated Arfs, including Arf1p, Arf2p, Arf3p, and Arl1p, have been shown to be associated with polarized budding, in which Arf1p, Arf2p, and Arl1p are involved in a secretion pathway through the Golgi network (40, 46). These secretory vesicles may be destined for the Spitzenkorper in filamentous fungi. In this study we found six Arf family proteins in A. nidulans. Three, namely, AN1126.3, AN5020.3, and AN5912.3 (ArfA, ArfB, and ArlA, respectively), are predicted to be myristoylated (31). Overexpression of ArfA has been shown to suppress the swoF myristoylation mutant (32). Here ArfB is investigated further, since it is the putative ortholog of Arf3p in S. cerevisiae associated with polarized bud site selection (22) and actin organization (11, 30). We show that ArfB is important for the establishment and maintenance of hyphal polarity, possibly by directing endocytosis in A. nidulans, and that this role requires N myristoylation of the protein.