Constitutive Activation of Endocytosis by Mutation ofmyoA, the Myosin I Gene of Aspergillus nidulans

Abstract

Class I myosins function in cell motility, intracellular vesicle trafficking and endocytosis. Recently, it was shown that class I myosins are phosphorylated by a member of the p21-activated kinase (PAK) family. PAK phosphorylates a conserved serine or threonine residue in the myosin heavy chain. Phosphorylation at this site is required for maximal activation of the actin-activated Mg 2؉ -ATPase activity in vitro. This serine or threonine residue is conserved in all known class I myosins of microbial origin and in the human and mouse class VI myosins. We have investigated the in vivo significance of this phosphorylation by mutating serine 371 of the class I myosin heavy chain gene myoA of Aspergillus nidulans. Mutation to glutamic acid, which mimics phosphorylation and therefore activation of the myosin, results in an accumulation of membranes in growing hyphae. This accumulation of membranes results from an activation of endocytosis. In contrast, mutation of serine 371 to alanine had no discernible effect on endocytosis. These studies are the first to demonstrate the in vivo significance of a regulatory phosphorylation on a class I myosin. Furthermore, our results suggest that MYOA has two functions, one dependent and one independent of phosphorylation.Class I myosins have been implicated in a variety of cellular processes, including cell locomotion, contractile vacuole function, receptor-mediated endocytosis, protein secretion, and intracellular vesicle transport (1). We have been studying the function of the class I myosin gene, myoA, of the filamentous fungus Aspergillus nidulans (2). We constructed a conditional null mutant myoA strain using the promoter of the inducible alcohol dehydrogenase gene alcA (2). Using this strain, we showed that myoA is an essential gene in A. nidulans and that it was required for the establishment of polarized hyphal growth. We also showed that strains lacking myoA were deficient for the secretion of acid phosphatase into the medium. In contrast, work on class I myosin function in the budding yeast Saccharomyces cerevisiae has suggested that they function in endocytosis and possibly protein secretion (3, 4). In S. cerevisiae there are two genes, MYO3 and MYO5, that code for class I myosin polypeptides. Deletion of either of these genes is not lethal, suggesting that they have overlapping or redundant functions. Deletion of both genes produces strains that either grow poorly (4) or are inviable (3). A strain that has a temperature-sensitive myo5 mutation is defective for endocytosis but not for protein secretion (3).Studies of class I myosin function and their actin-activated ATPase activity in Dictyostelium discoideum and Acanthamoeba castellanii have shown that these class I myosins are regulated by phosphorylation at a specific and highly conserved serine or threonine residue on the heavy chain (5, 6). This phosphorylation site is also found in other unconventional myosins like the class VI myosins from humans and mice (Fig. 1). Recent studies have shown that the kinase respon...

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)

“…Germination and hyphal branching are delayed, and growth rates are slower for both S371E and S371A mutant cells grown in liquid culture, but morphology and growth of both are normal on solid media (19). These results suggest either that, in contrast to A. castellanii myosin IC, these point mutations have little effect on the actin-dependent MgATPase activity of MYOA, or that the essential function(s) of MYOA may not require actin-dependent MgATPase activity.…”

“…It was surprising, therefore, to find that S371E and S371A mutants of MYOA are equally functional when expressed in A. nidulans lacking wild-type MYOA (19). Germination and hyphal branching are delayed, and growth rates are slower for both S371E and S371A mutant cells grown in liquid culture, but morphology and growth of both are normal on solid media (19).…”

The Tip Growth Apparatus ofAspergillus nidulans

“…Endocytosis of the fluorescent membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) requires actin (Penalva, 2005), and when FM4-64 is taken up, it initially localizes to organelles resembling actin patches. MYOA is important for endocytosis in A. nidulans, and GFP-MYOA localizes to cortical patches in vivo (Yamashita and May, 1998). Our data show that there is SYNA at the plasma membrane anterior to the actin/ABPA collar, but no SYNA at the plasma membrane posterior to the actin/ABPA collar.…”

Control of cell membrane tension by myosin-I

“…Experiments performed in multiple eukaryotic model systems have implicated class I myosins in various aspects of membranerelated events including phagocytosis (29)(30)(31)(32)(33), endocytosis (34)(35)(36)(37), exocytosis (38,39), and membrane recycling (40). Although our current data set does not allow us to rule out the possibility that perturbations in membrane trafficking may contribute to the changes in membrane tension observed in our experiments, our results do provide strong support for a model where class I myosins play a direct role in the control of membrane tension, by contributing to adhesion between the plasma membrane and underlying actin cytoskeleton.…”