Abstract
Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.
Highlights
20252 JOURNAL OF BIOLOGICAL CHEMISTRY membrane fusion [5, 6]
Identification of EHD2 as a Myoferlin Interacting Protein— Because myoferlin is abundantly expressed in prefusion myoblasts, C2C12 cells were grown to confluence in 10% fetal bovine serum to enrich for myoblasts in the early phases of fusion
We selected this protein for further analysis because EHD2 is a protein of the expected size that localizes in the cytoplasm and the membrane similar to myoferlin
Summary
20252 JOURNAL OF BIOLOGICAL CHEMISTRY membrane fusion [5, 6]. Because vesicle fusion is a common feature of most cells, it is possible that aspects of the myoblast fusion machinery utilize pathways more commonly used by other cell types for intracellular trafficking. These carboxyl-terminal EHD proteins are characterized by an amino-terminal nucleotide binding domain followed by a central coiled-coil domain [21] These carboxylterminal EHD proteins have been linked to endosomal trafficking including the recycling of cell surface receptors back to the plasma membrane [19, 22, 23]. Mutations in the presumed nucleotide binding domain of EHD, known as RME-1, lead to delay of recycling membrane components after endocytosis [24]. This role is conserved in mammals [25]. This work demonstrates that impaired endocytic recycling is associated with defective myoblast fusion
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