Bending a membrane: how clathrin affects budding.

Receptor-mediated endocytosis of ligands, such as transferrin and LDL, is suppressed when clathrin synthesis is blocked by RNA interference in HeLa cells. We have found that domains containing the adapter complex 2 (AP2)-coated vesicle adapter and the endocytic accessory proteins CALM (clathrin assembly lymphoid myeloid leukemia protein), epsin, and eps15/eps15R (EGF receptor pathway substrate 15-related) nevertheless persist at the plasma membrane. They are similar in size and number to those seen in clathrin-expressing cells.

Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation.

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the alpha-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane.

Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation.

Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation.