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Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling. [ABSTRACT FROM AUTHOR]

Arf GTPases regulate both the morphological and protein sorting events that are essential for membrane trafficking. Guanine nucleotide exchange factors (GEFs) specific for Arf proteins determine when and where Arf GTPases will be activated in cells. The yeast Gea2p Arf GEF is a member of an evolutionarily conserved family of high molecular mass Arf GEFs that are peripherally associated with membranes. Nothing is known about how these proteins are localized to membranes, and few direct binding partners have been identified. In yeast, Gea2p has been implicated in trafficking through the Golgi apparatus and in maintaining Golgi structure. A major function of the Golgi apparatus is the packaging of cargo into secretory granules or vesicles. This process occurs through a series of membrane transformation events starting with fenestration of a saccular membrane, and subsequent remodeling of the fenestrated membrane into a mesh-like tubular network. Concentration of secretory cargo into nodes of the tubular network leads to enlargement of the nodes, which correspond to forming vesicles/granules, and thinning of the surrounding tubules. The tubules eventually break to release the secretory vesicles/granules into the cytoplasm. This process is highly conserved at the morphological level from yeast to mammalian cells. Drs2p, a multi-span transmembrane domain protein and putative aminophospholipid translocase, is required for the formation of a class of secretory granules/vesicles in yeast. Here we show that Drs2p interacts directly with Gea2p, both in vitro and in vivo. We mapped the domain of interaction of Drs2p to a 20-amino-acid region of the Cterminal cytoplasmic tail of the protein, adjacent to a region essential for Drs2p function. Mutations in Gea2p that abolish interaction with Drs2p are clustered in the Cterminal third of the Sec7 domain, and are important for Gea2p function. We characterize one such mutant that has a thermosensitive phenotype, and show that it has morphological defects along the secretory pathway in the formation of secretory granules/vesicles. [ABSTRACT FROM AUTHOR]

P4-ATPases are transmembrane proteins unique to eukaryotes that play a fundamental role in vesicular transport. They have been proposed to act as phospholipid flippases thereby regulating lipid topology in cellular membranes. We cloned and characterized a novel murine P4-ATPase that is specifically expressed in testis, and named it FetA (flippase expressed in testis splicing form A). When expressed in Saccharomyces cerevisiae, FetA localizes partially to the plasma membrane resulting in increased internalization of NBD-labeled phosphatidylethanolamine and phosphatidylcholine, supporting a role for FetA in the inward lipid translocation across cellular membranes. In mouse testis, FetA protein is detected in gamete cells, from pachytene spermatocytes to mature sperms, and its intracellular localization is tightly related with acrosome formation, a process that involves intensive intracellular vesicle formation and fusion. Furthermore, loss-of-function of FetA by RNA interference in mastocytoma P815 cells profoundly perturbs the structural organization of the Golgi complex and causes loss of constitutive secretion at lower temperature. Our findings point to an essential role of FetA in Golgi morphology and secretory function, suggesting a crucial role for this novel murine P4-ATPase in spermatogenesis. [ABSTRACT FROM AUTHOR]