Abstract
The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI:http://dx.doi.org/10.7554/eLife.00190.001.
Highlights
Proteins on the surface of cells are removed from the plasma membrane by endocytosis
Clathrin-mediated endocytosis probably functions in all tissues, but it is unclear whether this process is suited to the high rates of endocytosis required at nerve terminals
The predominant mechanism for synaptic vesicle endocytosis is thought to be mediated via AP2 and clathrin (Dittman and Ryan, 2009)
Summary
Proteins on the surface of cells are removed from the plasma membrane by endocytosis. The endocytosis of clathrin-independent cargo hTAC is unaffected in both adaptin mutants (Figure 5—figure supplement 1, 2) Taken together, these data suggest that partially functional AP2 complexes might be present in mutations that eliminate single subunits. The skin-rescued worms have no detectable APA-2::GFP in the nervous system (Figure 3—figure supplement 1) and the skin promoter Pdpy-7 is only expressed in larval stages during development (Johnstone and Barry, 1996) These rescued animals are still egg-laying defective and slow-growing, but they provide an opportunity to study synaptic vesicle endocytosis in AP2-deficient synapses. In synapses of apa-2(ox422) apm-2(e840) double mutants (but rescued in the epidermis) the number of synaptic vesicles is reduced to 28% in acetylcholine neurons and 31% in GABA neurons (Figure 8B; Figure 8—figure supplement 1). The loss of both α- and μ2-adaptin leads to a more severe synaptic defect than the single mutants, suggesting that these subunits can function independently in synaptic vesicle endocytosis
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