title: Reduced plasma membrane expression of dysferlin mutants is attributed to accelerated endocytosis via a syntaxin-4-associated pathway.
abstract: Ferlins are an ancient family of C2 domain-containing proteins, with emerging roles in vesicular trafficking and human disease. Dysferlin mutations cause inherited muscular dystrophy, and dysferlin also shows abnormal plasma membrane expression in other forms of muscular dystrophy. We establish dysferlin as a short-lived (protein half-life approximately 4-6 h) and transitory transmembrane protein (plasma membrane half-life approximately 3 h), with a propensity for rapid endocytosis when mutated, and an association with a syntaxin-4 endocytic route. Dysferlin plasma membrane expression and endocytic rate is regulated by the C2B-FerI-C2C motif, with a critical role identified for C2C. Disruption of C2C dramatically reduces plasma membrane dysferlin (by 2.5-fold), due largely to accelerated endocytosis (by 2.5-fold). These properties of reduced efficiency of plasma membrane expression due to accelerated endocytosis are also a feature of patient missense mutant L344P (within FerI, adjacent to C2C). Importantly, dysferlin mutants that demonstrate accelerated endocytosis also display increased protein lability via endosomal proteolysis, implicating endosomal-mediated proteolytic degradation as a novel basis for dysferlin-deficiency in patients with single missense mutations. Vesicular labeling studies establish that dysferlin mutants rapidly transit from EEA1-positive early endosomes through to dextran-positive lysosomes, co-labeled by syntaxin-4 at multiple stages of endosomal transit. In summary, our studies define a transient biology for dysferlin, relevant to emerging patient therapeutics targeting dysferlin replacement. We introduce accelerated endosomal-directed degradation as a basis for lability of dysferlin missense mutants in dysferlinopathy, and show that dysferlin and syntaxin-4 similarly transit a common endosomal pathway in skeletal muscle cells.
authors.affiliation: Institute for Neuroscience and Muscle Research, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, New South Wales 2145, Australia.
authors.forename: Frances J
authors.initials: FJ
authors.lastname: Evesson
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Peat
authors.forename: Angela
authors.initials: A
authors.lastname: Lek
authors.forename: Fabienne
authors.initials: F
authors.lastname: Brilot
authors.forename: Harriet P
authors.initials: HP
authors.lastname: Lo
authors.forename: Russell C
authors.initials: RC
authors.lastname: Dale
authors.forename: Robert G
authors.initials: RG
authors.lastname: Parton
authors.forename: Kathryn N
authors.initials: KN
authors.lastname: North
authors.forename: Sandra T
authors.initials: ST
authors.lastname: Cooper
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D000073939:Dysferlin; D004705:Endocytosis; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D032342:Muscle Cells / Q000378:metabolism; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D018482:Muscle, Skeletal / Q000378:metabolism; D009136:Muscular Dystrophies / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D050765:Qa-SNARE Proteins